G2Cdb::Gene report

Gene id
Gene symbol
Homo sapiens
Rho-associated, coiled-coil containing protein kinase 2
G00000163 (Mus musculus)

Databases (7)

ENSG00000134318 (Ensembl human gene)
9475 (Entrez Gene)
515 (G2Cdb plasticity & disease)
ROCK2 (GeneCards)
604002 (OMIM)
Marker Symbol
HGNC:10252 (HGNC)
Protein Sequence
O75116 (UniProt)

Literature (48)

Pubmed - other

  • Distinct roles for ROCK1 and ROCK2 in the regulation of keratinocyte differentiation.

    Lock FE and Hotchin NA

    School of Biosciences, University of Birmingham, Birmingham, United Kingdom.

    Background: The human epidermis is comprised of several layers of specialized epithelial cells called keratinocytes. Normal homoeostasis of the epidermis requires that the balance between keratinocyte proliferation and terminal differentiation be tightly regulated. The mammalian serine/threonine kinases (ROCK1 and ROCK2) are well-characterised downstream effectors of the small GTPase RhoA. We have previously demonstrated that the RhoA/ROCK signalling pathway plays an important role in regulation of human keratinocyte proliferation and terminal differentiation. In this paper we addressed the question of which ROCK isoform was involved in regulation of keratinocyte differentiation.

    We used RNAi to specifically knockdown ROCK1 or ROCK2 expression in cultured human keratinocytes. ROCK1 depletion results in decreased keratinocyte adhesion to fibronectin and an increase in terminal differentiation. Conversely, ROCK2 depletion results in increased keratinocyte adhesion to fibronectin and inhibits terminal differentiation.

    Conclusion: These data suggest that ROCK1 and ROCK2 play distinct roles in regulating keratinocyte adhesion and terminal differentiation.

    Funded by: Cancer Research UK: C20692

    PloS one 2009;4;12;e8190

  • Evidence for Rho-kinase activation in patients with pulmonary arterial hypertension.

    Do e Z, Fukumoto Y, Takaki A, Tawara S, Ohashi J, Nakano M, Tada T, Saji K, Sugimura K, Fujita H, Hoshikawa Y, Nawata J, Kondo T and Shimokawa H

    Department of Cardiovascular Medicine, Tohoku University Graduate School of Medicine, Aoba-ku, Sendai, Japan.

    Background: Direct evidence for Rho-kinase activation in patients with pulmonary hypertension (PH) is still lacking.

    Rho-kinase activity in circulating neutrophils was examined by determining the ratio of phosphorylated/total forms of myosin-binding subunit, a substrate of Rho-kinase, in 40 consecutive PH patients and 40 healthy controls. Next, Rho-kinase expression and activity was examined in isolated human lung tissues (5 patients with idiopathic pulmonary arterial hypertension [IPAH], 5 controls) and vascular reactivity of isolated small human pulmonary arteries in vitro (4 IPAH, 4 controls). Rho-kinase activity in circulating neutrophils was significantly increased in the PH patients overall compared with controls (P<0.0001). Significant correlations were noted between Rho-kinase activity and the severity and duration of PAH (all P<0.05). Rho-kinase expression and activity in isolated lung tissues also were significantly increased in the IPAH patients compared with the controls (both P<0.0001). Endothelium-dependent relaxation was markedly impaired and serotonin-induced contraction (in the absence of the endothelium) markedly enhanced in the PAH patients compared with the controls, and the hypercontraction to serotonin was abolished by hydroxyfasudil, a specific Rho-kinase inhibitor.

    Conclusions: These results provide the first direct evidence for Rho-kinase activation in patients with PAH, suggesting the therapeutic importance of Rho-kinase in the disorder.

    Circulation journal : official journal of the Japanese Circulation Society 2009;73;9;1731-9

  • ROCK2 allelic variants are not associated with pre-eclampsia susceptibility in the Finnish population.

    Peterson H, Laivuori H, Kerkelä E, Jiao H, Hiltunen L, Heino S, Tiala I, Knuutila S, Rasi V, Kere J and Kivinen K

    Department of Biosciences and Nutrition, Karolinska Institutet, Stockholm, Sweden.

    The rho-associated coiled-coil protein kinase 2 (ROCK2) gene has been suggested to associate with general hypertension and is therefore a plausible functional candidate gene for pre-eclampsia. ROCK2 maps to chromosome 2p25, which we have implicated previously in a linkage study of pre-eclampsia. We have re-sequenced exons and putative promoter region of ROCK2 in up to 30 pre-eclampsia patients and 22 controls and genotyped putative functional single-nucleotide polymorphisms (SNPs) as well as tagging SNPs from HapMap in a Finnish case-control data set-340 affected and 357 matched control individuals-for a genetic association study of ROCK2 in pre-eclampsia. Even though several new SNPs were discovered, we did not detect significant allelic or haplotypic association between ROCK2 and pre-eclampsia. We assessed ROCK2 expression in placentas by microarray analysis, but no significant expression differences were observed when comparing preeclamptic and normotensive pregnancies. We conclude that common genetic variation in ROCK2 is unlikely to make a major contribution to the risk of pre-eclampsia, but cannot exclude the possibility of having missed non-coding functional variants or rare coding variants.

    Molecular human reproduction 2009;15;7;443-9

  • Protein kinase C activation disrupts epithelial apical junctions via ROCK-II dependent stimulation of actomyosin contractility.

    Ivanov AI, Samarin SN, Bachar M, Parkos CA and Nusrat A

    Department of Medicine, University of Rochester, Rochester, NY 14642, USA. Andrei_Ivanov@urmc.rochester.edu

    Background: Disruption of epithelial cell-cell adhesions represents an early and important stage in tumor metastasis. This process can be modeled in vitro by exposing cells to chemical tumor promoters, phorbol esters and octylindolactam-V (OI-V), known to activate protein kinase C (PKC). However, molecular events mediating PKC-dependent disruption of epithelial cell-cell contact remain poorly understood. In the present study we investigate mechanisms by which PKC activation induces disassembly of tight junctions (TJs) and adherens junctions (AJs) in a model pancreatic epithelium.

    Results: Exposure of HPAF-II human pancreatic adenocarcinoma cell monolayers to either OI-V or 12-O-tetradecanoylphorbol-13-acetate caused rapid disruption and internalization of AJs and TJs. Activity of classical PKC isoenzymes was responsible for the loss of cell-cell contacts which was accompanied by cell rounding, phosphorylation and relocalization of the F-actin motor nonmuscle myosin (NM) II. The OI-V-induced disruption of AJs and TJs was prevented by either pharmacological inhibition of NM II with blebbistatin or by siRNA-mediated downregulation of NM IIA. Furthermore, AJ/TJ disassembly was attenuated by inhibition of Rho-associated kinase (ROCK) II, but was insensitive to blockage of MLCK, calmodulin, ERK1/2, caspases and RhoA GTPase.

    Conclusion: Our data suggest that stimulation of PKC disrupts epithelial apical junctions via ROCK-II dependent activation of NM II, which increases contractility of perijunctional actin filaments. This mechanism is likely to be important for cancer cell dissociation and tumor metastasis.

    Funded by: NIDDK NIH HHS: DK 064399, DK 55679, DK 59888, DK 61379, DK 72564, R01 DK055679, R01 DK059888, R01 DK061379, R01 DK072564, R24 DK064399, R29 DK055679

    BMC cell biology 2009;10;36

  • Rho-kinase 2 is frequently overexpressed in hepatocellular carcinoma and involved in tumor invasion.

    Wong CC, Wong CM, Tung EK, Man K and Ng IO

    Department of Pathology, Liver Cancer and Hepatitis Research Laboratory, The University of Hong Kong, Hong Kong.

    Unlabelled: Deregulation of Rho family small guanosine triphosphatases has been implicated in human carcinogenesis. Rho-kinases are downstream effectors of Rho guanosine triphosphatases in the regulation of cytoskeletal reorganization and cell motility. However, their functions in human cancers remain elusive. In this study, we aimed to investigate the role of Rho-kinases in hepatocellular carcinoma (HCC) tumor progression and invasion. We first examined the expression of the two Rho-kinases (ROCK1 and ROCK2) in human HCC, and found that ROCK2 was frequently overexpressed in primary HCCs (22/41 [53.66%]). Clinico-pathological analysis revealed that overexpression of ROCK2 was significantly associated with the presence of tumor microsatellite formation (P = 0.005), suggesting that deregulation of ROCK2 may contribute to the intrahepatic metastasis of HCC. Consistently, we demonstrated that stable overexpression of ROCK2 significantly enhanced cell motility and invasiveness in HCC cells. Conversely, stable knockdown of ROCK2 by short hairpin RNA approach remarkably reduced HCC cell migration and invasion. Moreover, orthotopic liver xenograft models provided further support that stable knockdown of ROCK2 suppressed HCC invasion in vivo. Stable knockdown of ROCK2 in HCC cells significantly inhibited Golgi reorientation, myosin phosphatase phosphorylation, and formations of stress fibers, filopodia, and lamellipodia; these molecular and cellular events are crucial for cell motility and cancer invasion.

    Conclusion: Our results indicate that ROCK2 was overexpressed in human HCCs, and this overexpression was associated with a more aggressive biological behavior. Our findings also demonstrate that ROCK2 played a significant role in regulating cytoskeletal events and contributed to the invasion of HCC.

    Hepatology (Baltimore, Md.) 2009;49;5;1583-94

  • The hydrophobic motif of ROCK2 requires association with the N-terminal extension for kinase activity.

    Couzens AL, Saridakis V and Scheid MP

    Department of Biology, York University, 4700 Keele Street, Toronto, Ontario, Canada.

    ROCK (Rho-associated coiled-coil kinase) 2 is a member of the AGC kinase family that plays an essential role downstream of Rho in actin cytoskeleton assembly and contractility. The process of ROCK2 activation is complex and requires suppression of an autoinhibitory mechanism that is facilitated by Rho binding. ROCK2 harbours a C-terminal extension within the kinase domain that contains a hydrophobic cluster of phenylalanine and tyrosine residues surrounding a key threonine residue. In growth-factor-stimulated AGC kinases, the hydrophobic motif is important for the transition of the kinase from inactive to active complex and requires phosphorylation of the conserved serine/threonine residue. Less is understood about the contribution that the hydrophobic motif plays in the activation of ROCK, and the role of the hydrophobic motif threonine at position 405. In the present study, we show that this residue of ROCK is essential for substrate phosphorylation and kinase domain dimerization. However, in contrast with the growth-factor-activated AGC kinases, a phosphomimetic residue at position 405 was inhibitory for ROCK2 activity and dimerization. A soluble hydrophobic motif peptide allosterically activated ROCK2 In vitro, but not the equivalent peptide with Asp(405) substitution. Mechanistically, both ROCK2 activity and dimerization were dependent upon the interaction between Thr(405) of the hydrophobic motif and Asp(39) of the N-terminal extension. The reciprocal exchange of these residues was permissive for kinase activity, but dimerization was lost. These results support the rationale for development of small-molecule inhibitors designed to block ROCK activation by selectively interfering with hydrophobic motif-mediated activation-state transition and dimer formation.

    The Biochemical journal 2009;419;1;141-8

  • Inhibition of Rho-dependent kinases ROCK I/II activates VEGF-driven retinal neovascularization and sprouting angiogenesis.

    Kroll J, Epting D, Kern K, Dietz CT, Feng Y, Hammes HP, Wieland T and Augustin HG

    Center for Biomedicine and Medical Technology Mannheim, Joint Research Division Vascular Biology Medical Faculty Mannheim, Univ. of Heidelberg and the German Cancer Research Center, Ludolf-Krehl-Str. 13-17, 68167 Mannheim, Germany. kroll@angiogenese.de

    Vascular endothelial growth factor (VEGF) is an endothelial-specific growth factor that activates the small GTPase RhoA. While the role of RhoA for VEGF-driven endothelial migration and angiogenesis has been studied in detail, the function of its target proteins, the Rho-dependent kinases ROCK I and II, are controversially discussed. Using the mouse model of oxygen-induced proliferative retinopathy, ROCK I/II inhibition by H-1152 resulted in increased angiogenesis. This enhanced angiogenesis, however, was completely blocked by the VEGF-receptor antagonist PTK787/ZK222584. Loss-of-function experiments in endothelial cells revealed that inhibition of ROCK I/II using the pharmacological inhibitor H-1152 and ROCK I/II-specific small-interfering RNAs resulted in a rise of VEGF-driven sprouting angiogenesis. These functional data were biochemically substantiated by showing an enhanced VEGF-receptor kinase insert domain receptor phosphorylation and extracellular signal-regulated kinase 1/2 activation after inhibition of ROCK I/II. Thus our data identify that the inhibition of Rho-dependent kinases ROCK I/II activates angiogenesis both, in vitro and in vivo.

    American journal of physiology. Heart and circulatory physiology 2009;296;3;H893-9

  • ROCK isoform regulation of myosin phosphatase and contractility in vascular smooth muscle cells.

    Wang Y, Zheng XR, Riddick N, Bryden M, Baur W, Zhang X and Surks HK

    Molecular Cardiology Research Institute, Tufts Medical Center, Boston, MA 02111, USA.

    Abnormal vascular smooth muscle cell (VSMC) contraction plays an important role in vascular diseases. The RhoA/ROCK signaling pathway is now well recognized to mediate vascular smooth muscle contraction in response to vasoconstrictors by inhibiting myosin phosphatase (MLCP) activity and increasing myosin light chain phosphorylation. Two ROCK isoforms, ROCK1 and ROCK2, are expressed in many tissues, yet the isoform-specific roles of ROCK1 and ROCK2 in vascular smooth muscle and the mechanism of ROCK-mediated regulation of MLCP are not well understood. In this study, ROCK2, but not ROCK1, bound directly to the myosin binding subunit of MLCP, yet both ROCK isoforms regulated MLCP and myosin light chain phosphorylation. Despite that both ROCK1 and ROCK2 regulated MLCP, the ROCK isoforms had distinct and opposing effects on VSMC morphology and ROCK2, but not ROCK1, had a predominant role in VSMC contractility. These data support that although the ROCK isoforms both regulate MLCP and myosin light chain phosphorylation through different mechanisms, they have distinct roles in VSMC function.

    Funded by: NHLBI NIH HHS: HL074069, HL077378, P01 HL077378, P01 HL077378-05, R01 HL074069, R01 HL074069-05

    Circulation research 2009;104;4;531-40

  • Phosphorylated caveolin-1 regulates Rho/ROCK-dependent focal adhesion dynamics and tumor cell migration and invasion.

    Joshi B, Strugnell SS, Goetz JG, Kojic LD, Cox ME, Griffith OL, Chan SK, Jones SJ, Leung SP, Masoudi H, Leung S, Wiseman SM and Nabi IR

    Department of Cellular and Physiological Sciences, Life Sciences Institute, British Columbia Cancer Agency, Vancouver, British Columbia, Canada.

    Rho/ROCK signaling and caveolin-1 (Cav1) are implicated in tumor cell migration and metastasis; however, the underlying molecular mechanisms remain poorly defined. Cav1 was found here to be an independent predictor of decreased survival in breast and rectal cancer and significantly associated with the presence of distant metastasis for colon cancer patients. Rho/ROCK signaling promotes tumor cell migration by regulating focal adhesion (FA) dynamics through tyrosine (Y14) phosphorylation of Cav1. Phosphorylated Cav1 is localized to protrusive domains of tumor cells and Cav1 tyrosine phosphorylation is dependent on Src kinase and Rho/ROCK signaling. Increased levels of phosphorylated Cav1 were associated with elevated GTP-RhoA levels in metastatic tumor cells of various tissue origins. Stable expression and knockdown studies of Cav1 in tumor cells showed that phosphorylated Cav1 expression stimulates Rho activation, stabilizes FAK association with FAs, and promotes cell migration and invasion in a ROCK-dependent and Src-dependent manner. Tyrosine-phosphorylated Cav1, therefore, functions as an effector of Rho/ROCK signaling in the regulation of FA turnover and, thereby, tumor cell migration and invasion. These studies define a feedback loop between Rho/ROCK, Src, and phosphorylated Cav1 in tumor cell protrusions, identifying a novel function for Cav1 in tumor metastasis that may contribute to the poor prognosis of some Cav1-expressing tumors.

    Cancer research 2008;68;20;8210-20

  • The expression and prognostic value of ROCK I and ROCK II and their role in human breast cancer.

    Lane J, Martin TA, Watkins G, Mansel RE and Jiang WG

    Metastasis and Angiogenesis Research Group, University Department of Surgery, Wales College of Medicine, Cardiff University, Heath Park, Cardiff CF14 4XN, UK. lanej1@cf.ac.uk

    The role and expression of ROCKI and ROCKII in human breast cancer was investigated and the effect on clinical outcome assessed. ROCK knockdown cells (MDA-MB-231DeltaROCKI and MDA-MB-231DeltaROCKII were tested for their in vitro invasiveness, motility and in vivo tumour growth. Samples of fresh frozen breast tumour tissue (n=113) and normal background tissue (n=30) were processed for immunohistochemical and quantitative RT-PCR analyses. MDA-MB-231DeltaROCKI and MDA-MB-231DeltaROCKII cells showed significantly decreased invasiveness compared with control cells (mean +/- SEM 4.33+/-0.84 MDA-MB-231DeltaROCKI vs. 18.4+/-1.4 control, p<0.001; 6.8+/-1.2 MDA-MB-231DeltaROCKII vs. 18.4+/-1.4 control, p<0.001). Similarly, both exhibited reduced motility compared with control cells (p<0.001) and lost their response to HGF. Importantly, no significant difference existed between knockdown and control cells in in vivo tumour growth. ROCKI was significantly higher in human mammary tumours than normal background tissue (2.9+/-1.1 vs. 0.29+/-0.13, p=0.023), although expression of ROCKII was fairly consistent in both (2050+/-646 vs. 2303+/-2079). ROCKI expression was greater in patients who died from breast cancer than in patients who remained disease free (11.6+/-7.1 vs. 1.95+/-0.95). Higher levels of ROCKI were associated with increased grade (0.95+/-0.73 grade-1; 2.11+/-1.72 grade-2; and 4.06+/-1.99 grade-3). Levels of ROCKI, but not ROCKII, were significantly correlated with overall survival of patients (p=0.004, Univariate analysis, median follow-up 120 month). These results show that ROCKI and possibly ROCKII are key factors in regulation of motility/invasion of breast cancer cells. This, together with significant correlation between ROCKI and poor outcome in clinical breast cancer, indicates that it is a potential target in human breast cancer.

    International journal of oncology 2008;33;3;585-93

  • A major haplotype block at the rho-associated kinase 2 locus is associated with a lower risk of hypertension in a recessive manner: the HYPGENE study.

    Rankinen T, Church T, Rice T, Markward N, Blair SN and Bouchard C

    Human Genomics Laboratory, Pennington Biomedical Research Center, Baton Rouge, LA 70808, USA. rankint@pbrc.edu

    The goals of our study were to evaluate the haplotype pattern at the Rho-associated kinase 2 (ROCK2) locus and prospectively test the association between the ROCK2 haplotype-tagging single-nucleotide polymorphisms (tagSNPs) with hypertension for verified incident hypertensive patients (n=607) and healthy, normotensive controls (n=586) in a HYPGENE study. Rho-associated kinases (ROCKs) play a central role in signaling pathways that are involved in vascular smooth muscle contraction and endothelial nitric oxide availability. Using a set of stringent criteria (minor allele frequency>or=0.05, pairwise r2>or=0.95), we identified 18 tagSNPs from the 109 SNPs available in the HapMap Caucasian data set. TagSNPs were genotyped using the Illumina BeadStation platform. The 18 tagSNPs consisted of two linkage disequilibrium (LD) blocks. A haplotype defined by four SNPs (rs965665, rs10178332, rs6755196, rs10929732) in LD block 2 was recessively associated with a lower risk of hypertension (p=0.003). Homozygotes for the minor alleles had an 85% lower risk of hypertension than carriers of the common allele. The associations were independent of baseline age, cardiorespiratory fitness, body mass index, sex, and follow-up time. The LD block 2 spans about 137 kb of genomic DNA at the 5'-end of the ROCK2 locus and covers exons encoding the kinase domain of the protein. Our data strongly suggest that a major haplotype block at the ROCK2 locus is recessively associated with a lower risk of hypertension. Identification of functional mutation(s) could thus help in the development of ROCK2-specific treatments.

    Funded by: NHLBI NIH HHS: HL-069870, R01 HL069870, R01 HL069870-04; NIA NIH HHS: AG-06945, R01 AG006945, R37 AG006945

    Hypertension research : official journal of the Japanese Society of Hypertension 2008;31;8;1651-7

  • Rho-associated kinases are crucial for myofibroblast differentiation and production of extracellular matrix in scleroderma fibroblasts.

    Akhmetshina A, Dees C, Pileckyte M, Szucs G, Spriewald BM, Zwerina J, Distler O, Schett G and Distler JH

    University of Erlangen-Nuremberg, Erlangen, Germany.

    Objective: Rho-associated kinases (Rock) are the major cellular mediators of Rho GTPases and play an important role in the organization of the actin cytoskeleton. Inhibitors of Rock are currently being evaluated for the treatment of pulmonary arterial hypertension. This study was undertaken to analyze the role of Rock in the activation of fibroblasts in systemic sclerosis (SSc).

    Methods: Rock signaling was inhibited using chemical inhibitors and small interfering RNA. The expression of extracellular matrix (ECM) proteins and alpha-smooth muscle actin was analyzed by real-time polymerase chain reaction, Western blotting, and SirCol assay. Metabolic activity was quantified by MTT assay. Cell viability was assessed by staining with annexin V and propidium iodide. The role of MAP kinases was investigated using selective inhibitors and Western blotting.

    Results: Inhibition of Rock strongly reduced the synthesis of the major ECM proteins at the messenger RNA level as well as the protein level. Counterregulatory changes in the expression of tissue inhibitors of metalloproteinases and matrix metalloproteinases were not observed. Inhibition of Rock prevented myofibroblast differentiation. Transforming growth factor beta activated ERK in a Rock-dependent manner, and ERK mediated in part the stimulatory effects of Rock on myofibroblast differentiation. Toxic adverse effects of the inhibition of Rock were not observed.

    Conclusion: Our findings demonstrate that Rock potently stimulates the differentiation of resting fibroblasts into myofibroblasts and the production of ECM at biologically relevant concentrations without cell toxicity. These findings, along with the beneficial effects of Rock inhibition on vascular disease, indicate that inhibition of Rock might be an interesting novel therapeutic approach for the treatment of SSc.

    Arthritis and rheumatism 2008;58;8;2553-64

  • Rho kinases regulate corneal epithelial wound healing.

    Yin J and Yu FS

    Department of Ophthalmology, Kresge Eye Institute, Wayne State Univ. School of Medicine, 4717 St. Antoine Blvd, Detroit, MI, 48201, USA.

    We have previously shown that Rho small GTPase is required for modulating both cell migration and proliferation through cytoskeleton reorganization and focal adhesion formation in response to wounding. In the present study, we investigated the role of Rho kinases (ROCKs), major effectors of Rho GTPase, in mediating corneal epithelial wound healing. Both ROCK 1 and 2 were expressed and activated in THCE cells, an SV40-immortalized human corneal epithelial cell (HCEC) line, in response to wounding, lysophosphatidic acid, and heparin-binding EGF-like growth factor (HB-EGF) stimulations. The ROCK inhibitor Y-27632 efficiently antagonized ROCK activities without affecting Rho activation in wounded HCECs. Y-27632 promoted basal and HB-EGF-enhanced scratch wound healing and enhanced cell migration and adhesion to matrices, while retarded HB-EGF induced cell proliferation. E-cadherin- and beta-catenin-mediated cell-cell junction and actin cytoskeleton organization were disrupted by Y-27632. Y-27632 impaired the formation and maintenance of tight junction barriers indicated by decreased trans-epithelial resistance and disrupted occludin staining. We conclude that ROCK activities enhance cell proliferation, promote epithelial differentiation, but negatively modulate cell migration and cell adhesion and therefore play a role in regulating corneal epithelial wound healing.

    Funded by: NEI NIH HHS: NIH/NEI EY10869, R01 EY010869, R01 EY010869-13, R01 EY017960, R01 EY017960-01A2

    American journal of physiology. Cell physiology 2008;295;2;C378-87

  • Interactions among genetic variants from contractile pathway of vascular smooth muscle cell in essential hypertension susceptibility of Chinese Han population.

    Zhao Q, Wang L, Yang W, Chen S, Huang J, Fan Z, Li H, Lu X and Gu D

    Department of Evidence Based Medicine and Division of Population Genetics, Cardiovascular Institute and Fuwai Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, People's Republic of China.

    Recent study supports the hypothesis that the abnormalities of vascular smooth muscle cell (VSMC) that alter the intrinsic contractile state of the cell can directly cause abnormal vascular tone and disorders of blood pressure regulation, including hypertension. This study aimed to explore the individual and interactive effects of five genes from the contractile pathway of VSMC (KCNMB1, RGS2, PRKG, ROCK2, and MYLK) on the risk of essential hypertension.

    Methods: Potential functional polymorphisms of the five genes were analyzed in a large, representative Chinese Han sample of 4759 individuals, including 2411 hypertensive patients and 2348 age-matched and sex-matched healthy controls.

    Results: Single locus analyses showed significant association of the alleles of RGS2-rs34717272 with hypertension (original P of chi2 test=0.005; P value of permutation=0.019). After the adjustment for covariates, the carriers of minor D allele of RGS2-rs34717272 had an increased hypertension risk (DD+ID vs. II; odds ratio=1.19; 95% confidence interval, 1.04-1.35; P value after the Bonferroni correction=0.009 x 5=0.045). We also found that the carriers of minor T allele of KCNMB1-rs11739136 had a significantly decreased risk for hypertension (TT+CT vs. CC; odds ratio=0.83; 95% confidence interval, 0.72-0.95; P value after the Bonferroni correction=0.008 x 5=0.040). Final interaction models were selected and evaluated by permutation test and/or cross-validation test. Both the multifactor-dimensionality reduction and classification and regression trees methods showed a high-order gene-gene interaction among KCNMB1, RGS2, PRKG, and MYLK genes (P value of permutation in multifactor-dimensionality reduction=0.012).

    Conclusion: The overall results supported that the genetic variants in the contractile pathway of VSMC could contribute to hypertension risk independently or in an interactive manner.

    Pharmacogenetics and genomics 2008;18;6;459-66

  • Inhibition of ROCK activity allows InlF-mediated invasion and increased virulence of Listeria monocytogenes.

    Kirchner M and Higgins DE

    Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood, Boston, MA 02115, USA.

    Listeria monocytogenes is an intracellular bacterial pathogen that causes life-threatening disease. The mechanisms used by L. monocytogenes to invade non-professional phagocytic cells are not fully understood. In addition to the requirement of bacterial determinants, host cell conditions profoundly influence infection. Here, we have shown that inhibition of the RhoA/ROCK pathway by pharmacological inhibitors or RNA interference results in increased L. monocytogenes invasion of murine fibroblasts and hepatocytes. InlF, a member of the internalin multigene family with no known function, was identified as a L. monocytogenes-specific factor mediating increased host cell binding and entry. Conversely, activation of RhoA/ROCK activity resulted in decreased L. monocytogenes adhesion and invasion. Furthermore, virulence of wild-type bacteria during infection of mice was significantly increased upon inhibition of ROCK activity, whereas colonization and virulence of an inlF deletion mutant was not affected, thus supporting a role for InlF as a functional virulence determinant in vivo under specific conditions. In addition, inhibition of ROCK activity in human-derived cells enhanced either bacterial adhesion or adhesion and entry in an InlF-independent manner, further suggesting a host species or cell type-specific role for InlF and that additional bacterial determinants are involved in mediating ROCK-regulated invasion of human cells.

    Funded by: NIAID NIH HHS: AI53669, R01 AI053669, R01 AI053669-05

    Molecular microbiology 2008;68;3;749-67

  • Rho/Rho-associated kinase-II signaling mediates disassembly of epithelial apical junctions.

    Samarin SN, Ivanov AI, Flatau G, Parkos CA and Nusrat A

    Epithelial Pathobiology Research Unit, Department of Pathology and Laboratory Medicine, Emory University, Atlanta, GA 30322, USA.

    Apical junctional complex (AJC) plays a vital role in regulation of epithelial barrier function. Disassembly of the AJC is observed in diverse physiological and pathological states; however, mechanisms governing this process are not well understood. We previously reported that the AJC disassembly is driven by the formation of apical contractile acto-myosin rings. In the present study, we analyzed the signaling pathways regulating acto-myosin-dependent disruption of AJC by using a model of extracellular calcium depletion. Pharmacological inhibition analysis revealed a critical role of Rho-associated kinase (ROCK) in AJC disassembly in calcium-depleted epithelial cells. Furthermore, small interfering RNA (siRNA)-mediated knockdown of ROCK-II, but not ROCK-I, attenuated the disruption of the AJC. Interestingly, AJC disassembly was not dependent on myosin light chain kinase and myosin phosphatase. Calcium depletion resulted in activation of Rho GTPase and transient colocalization of Rho with internalized AJC proteins. Pharmacological inhibition of Rho prevented AJC disassembly. Additionally, Rho guanine nucleotide exchange factor (GEF)-H1 translocated to contractile F-actin rings after calcium depletion, and siRNA-mediated depletion of GEF-H1 inhibited AJC disassembly. Thus, our findings demonstrate a central role of the GEF-H1/Rho/ROCK-II signaling pathway in the disassembly of AJC in epithelial cells.

    Funded by: NIDDK NIH HHS: DK-055679, DK-59888, DK-61379, DK-72564, R01 DK055679, R01 DK059888, R01 DK061379, R01 DK072564, R29 DK055679

    Molecular biology of the cell 2007;18;9;3429-39

  • Proteomic screen defines the Polo-box domain interactome and identifies Rock2 as a Plk1 substrate.

    Lowery DM, Clauser KR, Hjerrild M, Lim D, Alexander J, Kishi K, Ong SE, Gammeltoft S, Carr SA and Yaffe MB

    Department of Biology, Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

    Polo-like kinase-1 (Plk1) phosphorylates a number of mitotic substrates, but the diversity of Plk1-dependent processes suggests the existence of additional targets. Plk1 contains a specialized phosphoserine-threonine binding domain, the Polo-box domain (PBD), postulated to target the kinase to its substrates. Using the specialized PBD of Plk1 as an affinity capture agent, we performed a screen to define the mitotic Plk1-PBD interactome by mass spectrometry. We identified 622 proteins that showed phosphorylation-dependent mitosis-specific interactions, including proteins involved in well-established Plk1-regulated processes, and in processes not previously linked to Plk1 such as translational control, RNA processing, and vesicle transport. Many proteins identified in our screen play important roles in cytokinesis, where, in mammalian cells, the detailed mechanistic role of Plk1 remains poorly defined. We go on to characterize the mitosis-specific interaction of the Plk1-PBD with the cytokinesis effector kinase Rho-associated coiled-coil domain-containing protein kinase 2 (Rock2), demonstrate that Rock2 is a Plk1 substrate, and show that Rock2 colocalizes with Plk1 during cytokinesis. Finally, we show that Plk1 and RhoA function together to maximally enhance Rock2 kinase activity in vitro and within cells, and implicate Plk1 as a central regulator of multiple pathways that synergistically converge to regulate actomyosin ring contraction during cleavage furrow ingression.

    Funded by: NCI NIH HHS: P50-CA112962, U54 CA112962; NIGMS NIH HHS: GM60594, R01 GM060594

    The EMBO journal 2007;26;9;2262-73

  • Diverse signaling pathways regulate fibroblast differentiation and transformation through Rho kinase activation.

    Harvey KA, Paranavitana CN, Zaloga GP and Siddiqui RA

    Cellular Biochemistry Laboratory, Methodist Research Institute, Clarian Health Partners, Indianapolis, Indiana, USA.

    This study examined the role of agonist-induced Rho kinase (ROCK) involvement in the morphological outcome of pulmonary-derived fibroblasts. Normal human lung fibroblasts (NHLF) spontaneously differentiate into network-like structures in a two-dimensional growth factor reduced Matrigel matrix-based assay. Sphingosine 1-phosphate (SPP), a bioactive phospholipid that regulates angiogenesis, inhibited fibroblast morphogenesis in a dose-dependent manner, virtually eliminating the presence of multi-cellular structures at 500 nM. Pretreatment with the Rho kinase-specific inhibitor, H1152, eradicated the high dose SPP-induced inhibition. Similarly, NHLFs transfected with Rho kinase siRNA prevented SPP-induced inhibition of the fibroblast morphogenesis. Alternatively, transforming growth factor-beta1 (TGF-beta1), a cytokine recognized as a key mediator of wound healing, terminally differentiates NHLF into myofibroblasts as evidenced by the expression of the smooth muscle cell isoform of alpha-actin (alpha-SMA). H1152 suppressed TGF-beta1-induced alpha-SMA expression in a dose-dependent manner. Similarly, treatment with Rho kinase siRNA reduced alpha-SMA expression by greater than 50%. SPP treatment had no effect on TGF-beta1-induced transformation into myofibroblasts, and TGF-beta1 treatment did not alter fibroblast morphogenesis. This study suggests a dual regulatory role for Rho kinase in cellular regulation of fibroblasts in which SPP-induced Rho kinase activation via a G-protein coupled receptor suppresses fibroblast morphogenesis while TGF-beta1-induced Rho kinase activation through a serine/threonine kinase receptor culminates in transformation into myofibroblasts.

    Journal of cellular physiology 2007;211;2;353-63

  • Notch1 is a p53 target gene involved in human keratinocyte tumor suppression through negative regulation of ROCK1/2 and MRCKalpha kinases.

    Lefort K, Mandinova A, Ostano P, Kolev V, Calpini V, Kolfschoten I, Devgan V, Lieb J, Raffoul W, Hohl D, Neel V, Garlick J, Chiorino G and Dotto GP

    Department of Biochemistry, University of Lausanne, Epalinges CH-1066, Switzerland.

    Little is known about the regulation and function of the Notch1 gene in negative control of human tumors. Here we show that Notch1 gene expression and activity are substantially down-modulated in keratinocyte cancer cell lines and tumors, with expression of this gene being under p53 control in these cells. Genetic suppression of Notch signaling in primary human keratinocytes is sufficient, together with activated ras, to cause aggressive squamous cell carcinoma formation. Similar tumor-promoting effects are also caused by in vivo treatment of mice, grafted with keratinocytes expressing oncogenic ras alone, with a pharmacological inhibitor of endogenous Notch signaling. These effects are linked with a lesser commitment of keratinocytes to differentiation, an expansion of stem cell populations, and a mechanism involving up-regulation of ROCK1/2 and MRCKalpha kinases, two key effectors of small Rho GTPases previously implicated in neoplastic progression. Thus, the Notch1 gene is a p53 target with a role in human tumor suppression through negative regulation of Rho effectors.

    Funded by: NCI NIH HHS: CA16038, CA73796, P01 CA016038, R01 CA073796; NIAMS NIH HHS: AR39190, R01 AR039190

    Genes & development 2007;21;5;562-77

  • Involvement of microtubules, p38, and Rho kinases pathway in 2-methoxyestradiol-induced lung vascular barrier dysfunction.

    Bogatcheva NV, Adyshev D, Mambetsariev B, Moldobaeva N and Verin AD

    University of Chicago, Department of Medicine, Chicago, Illinois 60637, USA.

    2-Methoxyestradiol (2ME), a promising anti-tumor agent, is currently tested in phase I/II clinical trial to assess drug tolerance and clinical effects. 2ME is known to affect microtubule (MT) polymerization rather than act through estrogen receptors. We hypothesized that 2ME, similar to other MT inhibitors, disrupts endothelial barrier properties. We show that 2ME decreases transendothelial electrical resistance and increases FITC-dextran leakage across human pulmonary artery endothelial monolayer, which correlates with 2ME-induced MT depolymerization. Pretreatment of endothelium with MT stabilizer taxol significantly attenuates the decrease in transendothelial resistance. 2ME treatment results in the induction of F-actin stress fibers, accompanied by the increase in myosin light chain (MLC) phosphorylation. The experiments with Rho kinase (ROCK) and MLC kinase inhibitors and ROCK small interfering RNA (siRNA) revealed that increase in MLC phosphorylation is attributed to the ROCK activation rather than MLC kinase activation. 2ME induces significant ERK1/2, p38, and JNK phosphorylation and activation; however, only p38 activation is relevant to the 2ME-induced endothelial hyperpermeability. p38 activation is accompanied by a marked increase in MAPKAP2 and 27-kDa heat shock protein (HSP27) phosphorylation level. Taxol significantly decreases p38 phosphorylation and activation in response to 2ME stimulation. Vice versa, p38 inhibitor SB203580 attenuates MT rearrangement in 2ME-challenged cells. Together, these results indicate that 2ME-induced barrier disruption is governed by MT depolymerization and p38- and ROCK-dependent mechanisms. The fact that certain concentrations of 2ME induce endothelial hyperpermeability suggests that the issue of the maximum-tolerated dose of 2ME for cancer treatment should be addressed with caution.

    Funded by: NHLBI NIH HHS: HL-067307, HL-083327, HL-58064, HL-80675

    American journal of physiology. Lung cellular and molecular physiology 2007;292;2;L487-99

  • Large-scale mapping of human protein-protein interactions by mass spectrometry.

    Ewing RM, Chu P, Elisma F, Li H, Taylor P, Climie S, McBroom-Cerajewski L, Robinson MD, O'Connor L, Li M, Taylor R, Dharsee M, Ho Y, Heilbut A, Moore L, Zhang S, Ornatsky O, Bukhman YV, Ethier M, Sheng Y, Vasilescu J, Abu-Farha M, Lambert JP, Duewel HS, Stewart II, Kuehl B, Hogue K, Colwill K, Gladwish K, Muskat B, Kinach R, Adams SL, Moran MF, Morin GB, Topaloglou T and Figeys D

    Protana, Toronto, Ontario, Canada.

    Mapping protein-protein interactions is an invaluable tool for understanding protein function. Here, we report the first large-scale study of protein-protein interactions in human cells using a mass spectrometry-based approach. The study maps protein interactions for 338 bait proteins that were selected based on known or suspected disease and functional associations. Large-scale immunoprecipitation of Flag-tagged versions of these proteins followed by LC-ESI-MS/MS analysis resulted in the identification of 24,540 potential protein interactions. False positives and redundant hits were filtered out using empirical criteria and a calculated interaction confidence score, producing a data set of 6463 interactions between 2235 distinct proteins. This data set was further cross-validated using previously published and predicted human protein interactions. In-depth mining of the data set shows that it represents a valuable source of novel protein-protein interactions with relevance to human diseases. In addition, via our preliminary analysis, we report many novel protein interactions and pathway associations.

    Molecular systems biology 2007;3;89

  • Physical interaction of two cancer-testis antigens, MAGE-C1 (CT7) and NY-ESO-1 (CT6).

    Cho HJ, Caballero OL, Gnjatic S, Andrade VC, Colleoni GW, Vettore AL, Outtz HH, Fortunato S, Altorki N, Ferrera CA, Chua R, Jungbluth AA, Chen YT, Old LJ and Simpson AJ

    Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021, USA.

    Cancer/testis (CT) antigens are the protein products of germ line-associated genes that are activated in a wide variety of tumors and can elicit autologous cellular and humoral immune responses. CT antigens can be divided between those that are encoded on the X chromosome (CT-X antigens) and those that are not (non-X CT antigens). Among the CT-X antigens, the melanoma antigen gene (MAGE) family, defined by a shared MAGE homology domain (MHD), is the largest. CT-X genes are frequently expressed in a coordinate manner in cancer cells, and their expression appears to be modulated by epigenetic mechanisms. The expression of CT-X genes is associated with advanced disease and poor outcome in different tumor types. We used the yeast two-hybrid system to identify putative MHD-interacting proteins. The MHD of MAGE-C1 (CT7) was used as bait to screen a human testis cDNA library. This study identified NY-ESO-1 (CT6) as a MAGE-C1 binding partner. Immunoprecipitation and immunofluorescence staining confirmed MAGE-C1 interaction with NY-ESO-1, and cytoplasmic co-localization of both proteins in melanoma cells. Co-expression of these two genes was found to occur in cancer cell lines from different origins, as well as in primary tumors (multiple myeloma and non-small cell lung cancer samples). This is the first report of direct interaction between two CT antigens and may be pertinent in the light of the frequently coordinated expression of these proteins.

    Cancer immunity 2006;6;12

  • Hypoxia-induced upregulation of endothelial small G protein RhoA and Rho-kinase/ROCK2 inhibits eNOS expression.

    Jin HG, Yamashita H, Nagano Y, Fukuba H, Hiji M, Ohtsuki T, Takahashi T, Kohriyama T, Kaibuchi K and Matsumoto M

    Department of Clinical Neuroscience and Therapeutics, Hiroshima University Graduate School of Biomedical Sciences, 1-2-3 Kasumi, Hiroshima 734-8551, Japan.

    The small G protein RhoA and its downstream effector Rho-kinase/ROCK2 play an important role in regulation of various vasculature cellular functions. Nitric oxide (NO) produced by endothelial NO synthase (eNOS) is an important mediator of vascular homeostasis and cerebral blood flow. Using the human endothelial cell line HUVEC, the present study investigated the role of RhoA and Rho-kinase in endothelial eNOS protein expression under hypoxic conditions as an in vitro model of ischemia. RhoA protein levels in HUVEC were low under normoxic conditions, but were significantly increased after 5h of hypoxia. Endothelial Rho-kinase expression was not detected until after 3h of hypoxia; such expression remained significantly increased after 5h. On the other hand, endothelial eNOS expression was similar after 3h of hypoxia, but was significantly decreased after 5h. The hypoxia-induced decrease in eNOS expression was significantly enhanced by expression of the constitutively active form of RhoA and significantly inhibited by suppression of RhoA expression by small interfering RNA. The hypoxia-induced decrease in eNOS expression was significantly inhibited when endogenous Rho-kinase activation was inhibited by Rho-binding domain expression. Furthermore, the hypoxia-induced decrease in eNOS expression was significantly enhanced by expression of the constitutively active form of Rho-kinase. Since expression and activation of RhoA and Rho-kinase inhibit eNOS expression in endothelial cells, attempts to down-regulate RhoA and Rho-kinase by multiple drugs, such as statins or Rho-kinase inhibitors, might provide endothelial and cardiovascular benefits through upregulation of eNOS.

    Neuroscience letters 2006;408;1;62-7

  • Global, in vivo, and site-specific phosphorylation dynamics in signaling networks.

    Olsen JV, Blagoev B, Gnad F, Macek B, Kumar C, Mortensen P and Mann M

    Center for Experimental BioInformatics, Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense, Denmark.

    Cell signaling mechanisms often transmit information via posttranslational protein modifications, most importantly reversible protein phosphorylation. Here we develop and apply a general mass spectrometric technology for identification and quantitation of phosphorylation sites as a function of stimulus, time, and subcellular location. We have detected 6,600 phosphorylation sites on 2,244 proteins and have determined their temporal dynamics after stimulating HeLa cells with epidermal growth factor (EGF) and recorded them in the Phosida database. Fourteen percent of phosphorylation sites are modulated at least 2-fold by EGF, and these were classified by their temporal profiles. Surprisingly, a majority of proteins contain multiple phosphorylation sites showing different kinetics, suggesting that they serve as platforms for integrating signals. In addition to protein kinase cascades, the targets of reversible phosphorylation include ubiquitin ligases, guanine nucleotide exchange factors, and at least 46 different transcriptional regulators. The dynamic phosphoproteome provides a missing link in a global, integrative view of cellular regulation.

    Cell 2006;127;3;635-48

  • A probability-based approach for high-throughput protein phosphorylation analysis and site localization.

    Beausoleil SA, Villén J, Gerber SA, Rush J and Gygi SP

    Department of Cell Biology, Harvard Medical School, 240 Longwood Ave., Boston, Massachusetts 02115, USA.

    Data analysis and interpretation remain major logistical challenges when attempting to identify large numbers of protein phosphorylation sites by nanoscale reverse-phase liquid chromatography/tandem mass spectrometry (LC-MS/MS) (Supplementary Figure 1 online). In this report we address challenges that are often only addressable by laborious manual validation, including data set error, data set sensitivity and phosphorylation site localization. We provide a large-scale phosphorylation data set with a measured error rate as determined by the target-decoy approach, we demonstrate an approach to maximize data set sensitivity by efficiently distracting incorrect peptide spectral matches (PSMs), and we present a probability-based score, the Ascore, that measures the probability of correct phosphorylation site localization based on the presence and intensity of site-determining ions in MS/MS spectra. We applied our methods in a fully automated fashion to nocodazole-arrested HeLa cell lysate where we identified 1,761 nonredundant phosphorylation sites from 491 proteins with a peptide false-positive rate of 1.3%.

    Funded by: NHGRI NIH HHS: HG03456; NIGMS NIH HHS: GM67945

    Nature biotechnology 2006;24;10;1285-92

  • Rho kinase polymorphism influences blood pressure and systemic vascular resistance in human twins: role of heredity.

    Seasholtz TM, Wessel J, Rao F, Rana BK, Khandrika S, Kennedy BP, Lillie EO, Ziegler MG, Smith DW, Schork NJ, Brown JH and O'Connor DT

    Department of Pharmacology, University of California, San Diego, CA, USA.

    The Rho/Rho kinase (ROCK) pathway is implicated in experimental hypertension. We, therefore, explored the role of ROCK2 genetic variation in human blood pressure (BP) regulation, exploiting the advantages of a human twin sample to probe heritability. The focus of this work is the common nonsynonymous variant at ROCK2: Thr431Asn. Cardiovascular and autonomic traits displayed substantial heritability (from approximately 33% to 71%; P<0.05). The Asn/Asn genotype (compared with Asn/Thr or Thr/Thr) was associated with greater resting systolic (P<0.001), diastolic (P<0.0001), and mean BP (P<0.0001); allelic variation at ROCK2 accounted for up to approximately 5% of BP variation (P<0.0001). Systemic vascular resistance was higher in Asn/Asn individuals (P=0.049), whereas cardiac output, large artery compliance, and vasoactive hormone secretion were not different. Coupling of the renin-angiotensin system to systemic resistance and BP was diminished in Asn/Asn homozygotes, suggesting genetic pleiotropy of Thr431Asn, confirmed by bivariate genetic analyses. The Asn/Asn genotype also predicted higher BP after environmental (cold) stress. The rise in heart rate after cold was less pronounced in Asn/Asn individuals, consistent with intact baroreceptor function, and baroreceptor slope was not influenced by genotype. Common genetic variation (Thr431Asn) at ROCK2 predicts increased BP, systemic vascular resistance (although not large artery compliance), and resistance in response to the endogenous renin-angiotensin system, indicating a resistance vessel-based effect on elevated BP. The results suggest that common variation in ROCK2 exerts systemic resistance-mediated changes in BP, documenting a novel mechanism for human circulatory control, and suggesting new possibilities for diagnostic profiling and treatment of subjects at risk of developing hypertension.

    Funded by: NCRR NIH HHS: M01 RR000827, M01 RR000827-346233; NHLBI NIH HHS: L30 HL110303, P01 HL058120, P01 HL058120-10, P01 HL058120-100004, P01 HL058120-109006

    Hypertension (Dallas, Tex. : 1979) 2006;47;5;937-47

  • Molecular mechanism for the regulation of rho-kinase by dimerization and its inhibition by fasudil.

    Yamaguchi H, Kasa M, Amano M, Kaibuchi K and Hakoshima T

    Structural Biology Laboratory, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan.

    Rho-kinase is a key regulator of cytoskeletal events and a promising drug target in the treatment of vascular diseases and neurological disorders. Unlike other protein kinases, Rho-kinase requires both N- and C-terminal extension segments outside the kinase domain for activity, although the details of this requirement have been elusive. The crystal structure of an active Rho-kinase fragment containing the kinase domain and both the extensions revealed a head-to-head homodimer through the N-terminal extension forming a helix bundle that structurally integrates the C-terminal extension. This structural organization enables binding of the C-terminal hydrophobic motif to the N-terminal lobe, which defines the correct disposition of helix alphaC that is important for the catalytic activity. The bound inhibitor fasudil significantly alters the conformation and, consequently, the mode of interaction with the catalytic cleft that contains local structural changes. Thus, both kinase and drug conformational pliability and stability confer selectivity.

    Structure (London, England : 1993) 2006;14;3;589-600

  • Screening for cell migration inhibitors via automated microscopy reveals a Rho-kinase inhibitor.

    Yarrow JC, Totsukawa G, Charras GT and Mitchison TJ

    Department of Systems Biology, Boston, Massachusetts 02115, USA. jyarrow@post.harvard.edu

    Small-molecule kinase inhibitors are predominantly discovered in pure protein assays. We have discovered an inhibitor of Rho-kinase (ROCK) through an image-based, high-throughput screen of cell monolayer wound healing. Using automated microscopy, we screened a library of approximately 16,000 compounds finding many that affected cell migration or cell morphology as well as compounds that blocked mitotic progression. We tested approximately 200 compounds in a series of subassays and chose one, 3-(4-pyridyl)indole (Rockout), for more detailed characterization. Rockout inhibits blebbing and causes dissolution of actin stress fibers, phenocopying Rho-kinase inhibitors. Testing Rho-kinase activity in vitro, Rockout inhibits with an IC50 of 25 microM ( approximately 5-fold less potent than Y-27632) but has a similar specificity profile. We also profile the wound healing assay with a library of compounds with known bioactivities, revealing multiple pathways involved in the biology.

    Funded by: NIGMS NIH HHS: GM62566

    Chemistry & biology 2005;12;3;385-95

  • Direct cleavage of ROCK II by granzyme B induces target cell membrane blebbing in a caspase-independent manner.

    Sebbagh M, Hamelin J, Bertoglio J, Solary E and Bréard J

    Institut National de la Santé et de la Recherche Medicale (INSERM) U461, IFR75, Faculté de Pharmacie, 92296 Châtenay-Malabry, France.

    Caspase activation in target cells is a major function of granzyme B (grB) during cytotoxic lymphocyte granule-induced apoptosis. grB-mediated cell death can occur in the absence of active caspases, and the molecular targets responsible for this additional pathway remain poorly defined. Apoptotic plasma membrane blebbing is caspase independent during granule exocytosis-mediated cell death, whereas in other instances, this event is a consequence of the cleavage by caspases of the Rho effector, Rho-associated coiled coil-containing protein kinase (ROCK) I. We show here that grB directly cleaves ROCK II, a ROCK family member encoded by a separate gene and closely related to ROCK I, and this causes constitutive kinase activity and bleb formation. For the first time, two proteins of the same family are found to be specifically cleaved by either a caspase or grB, thus defining two independent pathways with similar phenotypic consequences in the cells. During granule-induced cell death, ROCK II cleavage by grB would overcome, for this apoptotic feature, the consequences of deficient caspase activation that may occur in virus-infected or malignant target cells.

    The Journal of experimental medicine 2005;201;3;465-71

  • Large-scale characterization of HeLa cell nuclear phosphoproteins.

    Beausoleil SA, Jedrychowski M, Schwartz D, Elias JE, Villén J, Li J, Cohn MA, Cantley LC and Gygi SP

    Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.

    Determining the site of a regulatory phosphorylation event is often essential for elucidating specific kinase-substrate relationships, providing a handle for understanding essential signaling pathways and ultimately allowing insights into numerous disease pathologies. Despite intense research efforts to elucidate mechanisms of protein phosphorylation regulation, efficient, large-scale identification and characterization of phosphorylation sites remains an unsolved problem. In this report we describe an application of existing technology for the isolation and identification of phosphorylation sites. By using a strategy based on strong cation exchange chromatography, phosphopeptides were enriched from the nuclear fraction of HeLa cell lysate. From 967 proteins, 2,002 phosphorylation sites were determined by tandem MS. This unprecedented large collection of sites permitted a detailed accounting of known and unknown kinase motifs and substrates.

    Funded by: NHGRI NIH HHS: HG00041, K22 HG000041, T32 HG000041; NIGMS NIH HHS: GM67945, GMS6203, R01 GM056203, R01 GM067945

    Proceedings of the National Academy of Sciences of the United States of America 2004;101;33;12130-5

  • Remnant lipoproteins from patients with sudden cardiac death enhance coronary vasospastic activity through upregulation of Rho-kinase.

    Oi K, Shimokawa H, Hiroki J, Uwatoku T, Abe K, Matsumoto Y, Nakajima Y, Nakajima K, Takeichi S and Takeshita A

    Department of Cardiovascular Medicine, Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan.

    Objective: Sudden cardiac death (SCD) still remains a serious problem. We have previously shown that remnant-like particles (RLP) are the major risk factor for SCD and that Rho-kinase plays a central role in the molecular mechanism of coronary vasospasm. In this study, we examined whether RLP from patients with SCD upregulate Rho-kinase associated with an enhanced coronary vasospastic activity.

    We isolated RLP and non-RLP in very-low-density lipoprotein (VLDL) fraction from SCD patients without coronary stenosis. We performed in vivo study in which we treated the coronary artery with RLP or non-RLP fraction at the adventitia in pigs. After 1 week, intracoronary serotonin caused marked coronary hyperconstriction at the segment treated with RLP fraction but not with non-RLP fraction (P<0.001, n=6), and hydroxyfasudil, a selective Rho-kinase inhibitor, dose-dependently inhibited the spasm in vivo. In organ chamber experiments, serotonin caused hypercontraction of vascular smooth muscle cells (VSMC) from RLP-treated segment, which was significantly inhibited by hydroxyfasudil (P<0.001, n=6). In cultured human coronary VSMC, the treatment with RLP significantly enhanced the expression and activity of Rho-kinase (P<0.05, n=6).

    Conclusions: These results indicate that RLP from SCD patients upregulate Rho-kinase in coronary VSMC and markedly enhance coronary vasospastic activity.

    Arteriosclerosis, thrombosis, and vascular biology 2004;24;5;918-22

  • The composition of Staufen-containing RNA granules from human cells indicates their role in the regulated transport and translation of messenger RNAs.

    Villacé P, Marión RM and Ortín J

    Centro Nacional de Biotecnología, Campus de Cantoblanco, 28049 Madrid, Spain.

    hStaufen is the human homolog of dmStaufen, a double-stranded (ds)RNA-binding protein involved in early development of the fly. hStaufen-containing complexes were purified by affinity chromatography from human cells transfected with a TAP-tagged hStaufen gene. These complexes showed a size >10 MDa. Untagged complexes with similar size were identified from differentiated human neuroblasts. The identity of proteins present in purified hStaufen complexes was determined by mass spectrometry and the presence of these proteins and other functionally related ones was verified by western blot. Ribosomes and proteins involved in the control of protein synthesis (PABP1 and FMRP) were present in purified hStaufen complexes, as well as elements of the cytoskeleton (tubulins, tau, actin and internexin), cytoskeleton control proteins (IQGAP1, cdc42 and rac1) and motor proteins (dynein, kinesin and myosin). In addition, proteins normally found in the nucleus, like nucleolin and RNA helicase A, were also found associated with cytosolic hStaufen complexes. The co-localization of these components with hStaufen granules in the dendrites of differentiated neuroblasts, determined by confocal immunofluorescence, validated their association in living cells. These results support the notion that the hStaufen-containing granules are structures essential in the localization and regulated translation of human mRNAs in vivo.

    Nucleic acids research 2004;32;8;2411-20

  • Activation of RhoA and inhibition of myosin phosphatase as important components in hypertension in vascular smooth muscle.

    Seko T, Ito M, Kureishi Y, Okamoto R, Moriki N, Onishi K, Isaka N, Hartshorne DJ and Nakano T

    First Department of Internal Medicine, Mie University School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507, Japan.

    Two mechanisms are proposed to account for the inhibition of myosin phosphatase (MP) involved in Ca2+ sensitization of vascular muscle, ie, phosphorylation of either MYPT1, a target subunit of MP or CPI-17, an inhibitory phosphoprotein. In cultured vascular aorta smooth muscle cells (VSMCs), stimulation with angiotensin II activated RhoA, and this was blocked by pretreatment with 8-bromo-cGMP. VSMCs stimulated by angiotensin II, endothelin-1, or U-46619 significantly increased the phosphorylation levels of both MYPT1 (at Thr696) and CPI-17 (at Thr38). The angiotensin II-induced phosphorylation of MYPT1 was completely blocked by 8-bromo-cGMP or Y-27632 (a Rho-kinase inhibitor), but not by GF109203X (a PKC inhibitor). In contrast, phosphorylation of CPI-17 was inhibited only by GF109203X. Y-27632 dramatically corrected the hypertension in N(omega)-nitro-L-arginine methyl ester (L-NAME)-treated rats, and this hypertension also was sensitive to isosorbide mononitrate. The level of the active form of RhoA was significantly higher in aortas from L-NAME-treated rats. Expression of RhoA, Rho-kinase, MYPT1, CPI-17, and myosin light chain kinase were not significantly different in aortas from L-NAME-treated and control rats. Activation of RhoA without changes in levels of other signaling molecules were observed in three other rat models of hypertension, ie, stroke-prone spontaneously hypertensive rats, renal hypertensive rats, and DOCA-salt rats. These results suggest that independent of the cause of hypertension, a common point in downstream signaling and a critical component of hypertension is activation of RhoA and subsequent activation of Rho-kinase.

    Funded by: NHLBI NIH HHS: HL23615

    Circulation research 2003;92;4;411-8

  • Phosphorylation of the myosin phosphatase targeting subunit and CPI-17 during Ca2+ sensitization in rabbit smooth muscle.

    Kitazawa T, Eto M, Woodsome TP and Khalequzzaman M

    Department of Physiology and Biophysics, Georgetown University Medical Center, Washington, DC 20007, USA. kitazawa@bbri.org

    Myosin phosphatase (MLCP) plays a critical regulatory role in the Ca(2+) sensitivity of myosin phosphorylation and smooth muscle contraction. It has been suggested that phosphorylation at Thr(695) of the MLCP regulatory subunit (MYPT1) and at Thr(38) of the MLCP inhibitor protein CPI-17 results in inhibition of MLCP activity. We have previously demonstrated that CPI-17 Thr(38) phosphorylation plays an important role in G-protein-mediated inhibition of MLCP in tonic arterial smooth muscle. Here, we attempted to evaluate the function of MYPT1 in phasic rabbit portal vein (PV) and vas deferens (VD) smooth muscles. Using site- and phospho-specific antibodies, phosphorylation of MYPT1 Thr(695) and CPI-17 Thr(38) was examined along with MYPT1 Thr(850), which is a non-inhibitory Rho-kinase site. We found that both CPI-17 Thr(38) and MYPT1 Thr(850) were phosphorylated in response to agonists or GTPgammaS concurrently with contraction and myosin phosphorylation in alpha-toxin-permeabilized PV tissues. In contrast, phosphorylation of MYPT1 Thr(695) did not increase. Comparable results were also obtained in both permeabilized and intact VD. The Rho-kinase inhibitor Y-27632 and the protein kinase C (PKC) inhibitor GF109203X suppressed phosphorylation of MYPT1 Thr(850) and CPI-17 Thr(38), respectively, in intact VD while MYPT1 Thr(695) phosphorylation was insensitive to both inhibitors. These results indicate that phosphorylation of MYPT1 Thr(695) is independent of stimulation of G-proteins, Rho-kinase or PKC. In the phasic PV, phosphorylation of CPI-17 Thr(38) may contribute towards inhibition of MLCP while the phasic visceral VD, which has a low CPI-17 concentration, probably utilizes other Ca(2+) sensitizing mechanisms for inhibiting MLCP besides phosphorylation of MYPT1 and CPI-17.

    Funded by: NHLBI NIH HHS: HL70881, R01 HL070881, R01HL51824

    The Journal of physiology 2003;546;Pt 3;879-89

  • p80 ROKalpha binding protein is a novel splice variant of CRMP-1 which associates with CRMP-2 and modulates RhoA-induced neuronal morphology.

    Leung T, Ng Y, Cheong A, Ng CH, Tan I, Hall C and Lim L

    Glaxo-IMCB Group, Institute of Molecular and Cell Biology, 30 Medical Drive, 117609, Singapore. mcbthoml@imcb.nus.edu.sg

    Using antibody against the Rho binding domain of ROKalpha, two neuronal phosphoproteins of 62 and 80 kDa were co-immunoprecipitated from brain extracts. Peptide analysis revealed their identity as collapsin response mediator proteins (CRMPs); p62 was CRMP-2 whereas p80 was a novel splice form of CRMP-1 with an extended N-terminus. p80 CRMP-1 was able to complex with CRMP-2, suggesting that p80 CRMP-1 and CRMP-2 form oligomers. CRMP-2 was the major substrate of ROK. p80 CRMP-1 interacted with the kinase domain of ROKalpha, resulting in inhibition of the catalytic activity towards other substrates. Over-expression of p80 CRMP-1 and CRMP-2 together counteracted the effects of RhoA on neurite retraction, an effect enhanced by mutation of the ROK phosphorylation site in CRMP-2. p80 CRMP-1 and CRMP-2 may be modulators of RhoA-dependent signaling, through interaction with and regulation of ROKalpha.

    FEBS letters 2002;532;3;445-9

  • Phosphorylation of the regulatory subunit of smooth muscle protein phosphatase 1M at Thr850 induces its dissociation from myosin.

    Velasco G, Armstrong C, Morrice N, Frame S and Cohen P

    Division of Signal Transduction Therapy, School of Life Sciences, University of Dundee, DD1 5EH, Dundee, UK.

    Rho kinase is known to control smooth muscle contractility by phosphorylating the 110 kDa myosin-targetting subunit (MYPT1) of the myosin-associated form of protein phosphatase 1 (PP1M). Phosphorylation of MYPT1 at Thr695 has previously been reported to inhibit the catalytic activity of PP1. Here, we show that the phosphorylation of Thr850 by Rho kinase dissociates PP1M from myosin, providing a second mechanism by which myosin phosphatase activity is inhibited.

    FEBS letters 2002;527;1-3;101-4

  • Characterization and purification of truncated human Rho-kinase II expressed in Sf-21 cells.

    Turner MS, Fen-Fen-Lin, Trauger JW, Stephens J and LoGrasso P

    Department of Molecular Neuroscience, Merck Research Laboratories, 3535 General Atomics Court, MRLSDB1, San Diego, CA 92121, USA.

    Rho-kinase II (ROCK-II) is a serine/threonine kinase that is involved in regulation of smooth muscle contraction and has been shown to contribute to the early stages of axon formation in neurons and the regulation of the neuronal cytoskeleton. Much of what is known about Rho-kinase function comes from cell-biological studies, whereas a paucity of biochemical characterization exists for the enzyme. In an effort to characterize ROCK-II biochemically we have cloned a truncated form of human ROCK-II comprising amino acids 1-543 and overexpressed it in Sf-21 cells. Utilizing the Sf-21/baculovirus expression system we isolated milligram quantities of ROCK-II (1-543) and purified the enzyme to near homogeneity. Optimal expression conditions revealed that infection of Sf-21 cells at a multiplicity of infection of 10 for 72h yielded maximal protein expression. Expression of ROCK-II (1-543) as an N-terminal Flag fusion protein allowed a single-step purification yielding greater than 90% homogeneous protein as assessed by SDS-PAGE. Enzyme activity was linear over a range of enzyme concentrations and times. Capture of phosphorylated, biotinylated peptides on streptavidin membrane allowed assessment of peptide substrate preference and measurement of steady-state rate constants. The data indicated that an 11-mer peptide containing Ser235/Ser236 of the S6 ribosomal protein and a 12-mer peptide containing Thr508 of LIM kinase were preferred substrates for ROCK-II (1-543). Finally, staurosporine had an IC(50) value 215-fold more potent than that of the ROCK inhibitor Y-27632. Collectively these data lay the foundation for the beginning of a biochemical characterization for this enzyme and provide methodology for more detailed biochemical, biophysical, and kinetic analysis.

    Archives of biochemistry and biophysics 2002;405;1;13-20

  • Rho-kinase contributes to diphosphorylation of myosin II regulatory light chain in nonmuscle cells.

    Ueda K, Murata-Hori M, Tatsuka M and Hosoya H

    Department of Biological Science, Graduate School of Science, Hiroshima University, Higashi-Hiroshima, 739-8526, Japan.

    Phosphorylation of myosin II regulatory light chain (MRLC) is important for cell motility and cytokinesis in nonmuscle cells. Although the regulation of monophosphorylated MRLC at serine 19 throughout the cell cycle was examined in detail, MRLC diphosphorylation at both threonine 18 and serine 19 is still unclear. Here we found that Rho-kinase has an activity for MRLC diphosphorylation in nonmuscle cells using sequential column chromatographies. Transfection of Rho-kinase-EGFP induced the excess diphosphorylated MRLC and the bundling of the actin filaments. Conversely, the treatment of cells with a specific inhibitor of Rho-kinase, Y-27632, resulted in the decrease of endogenous diphosphorylated MRLC and actin stress fibers. Immunolocalization studies showed that both diphosphorylated MRLC and Rho-kinase accumulated and colocalized at the contractile ring and the midbody in dividing cells. Taken together, it is suggested that Rho-kinase contributes to MRLC diphosphorylation and reorganization of actin filaments in nonmuscle cells.

    Oncogene 2002;21;38;5852-60

  • ROCK-II-induced membrane blebbing and chromatin condensation require actin cytoskeleton.

    Song Y, Hoang BQ and Chang DD

    Department of Medicine, Microbiology, Immunology, and Molecular Genetics, UCLA School of Medicine, Los Angeles, California 900095-1678, USA.

    Ectopic expression of ROCK II (Rho kinase II or ROKalpha), an effector of Rho GTPase, induces membrane blebbing and chromatin condensation. ROCK II can induce membrane blebbing in the presence of the caspase inhibitor z-VAD-fmk or in caspase-3-deficient MCF-7 cells, indicating that the activation of caspases is not required. ROCK-II-induced membrane blebbing, however, is reversed by the myosin light chain kinase inhibitor ML-7 or cytochalasin D. In addition, the expression of a constitutively activated form of cofilin (S3A-cofilin) suppresses both membrane blebbing and chromatin condensation in ROCK II expressing cells. These findings suggest that the activation of actin-myosin contractility is responsible for membrane blebbing and chromatin condensation and implicate ROCK II as a potential mediator of the morphological changes associated with apoptosis.

    Funded by: NCI NIH HHS: CA78375; NIDCR NIH HHS: T32DE007296

    Experimental cell research 2002;278;1;45-52

  • Kinetic mechanism for human Rho-Kinase II (ROCK-II).

    Trauger JW, Lin FF, Turner MS, Stephens J and LoGrasso PV

    Department of Molecular Neuroscience, Merck Research Laboratories, 3535 General Atomics Court, San Diego, CA 92121, USA.

    Rho-Kinase is a serine/threonine kinase that is involved in the regulation of smooth muscle contraction and cytoskeletal reorganization of nonmuscle cells. While the signal transduction pathway in which Rho-Kinase participates has been and continues to be extensively studied, the kinetic mechanism of Rho-Kinase-catalyzed phosphorylation has not been investigated. We report here elucidation of the kinetic mechanism for Rho-Kinase by using steady-state kinetic studies. These studies used the kinase domain of human Rho-Kinase II (ROCK-II 1-534) with S6 peptide (biotin-AKRRRLSSLRA-NH(2)) as the phosphorylatable substrate. Double-reciprocal plots for two-substrate kinetic data yielded intersecting line patterns with either ATP or S6 peptide as the varied substrate, indicating that Rho-Kinase utilized a ternary complex (sequential) kinetic mechanism. Dead-end inhibition studies were used to investigate the order of binding for ATP and the peptide substrate. The ATP-competitive inhibitors AMP-PCP and Y-27632 were noncompetitive inhibitors versus S6 peptide, and the S6 peptide analogue S6-AA (acetyl-AKRRRLAALRA-NH(2)) was a competitive inhibitor versus S6 peptide and a noncompetitive inhibitor versus ATP. These results indicated a random order of binding for ATP and S6 peptide.

    Biochemistry 2002;41;28;8948-53

  • Integrin-linked kinase phosphorylates the myosin phosphatase target subunit at the inhibitory site in platelet cytoskeleton.

    Kiss E, Murányi A, Csortos C, Gergely P, Ito M, Hartshorne DJ and Erdodi F

    Department of Medical Chemistry, University of Debrecen, Medical and Health Science Center, H-4026 Debrecen, Bem tér 18/B, Hungary.

    The myosin phosphatase (MP) composed of the catalytic subunit of type 1 protein phosphatase and myosin phosphatase target subunit isoform 1 (MYPT1) was identified as the major serine/threonine phosphatase component in the platelet-cytoskeleton fraction. MYPT1 was phosphorylated by cytoskeletal kinase(s), but the identity of the kinase(s) and the effect of phosphorylation were not established. Incubation of platelet-cytoskeletal fraction with MgATP or MgATP[S] (magnesium adenosine 5'-[gamma-thio]triphosphate) caused a decrease in the 20 kDa light-chain of smooth-muscle myosin (MLC20) phosphatase and phosphorylase phosphatase activities. MYPT1 contains a phosphorylation site, Thr-695, involved in the inhibition of MP in a RhoA/Rho kinase-dependent manner. The cytoskeletal kinase(s) phosphorylated Thr-695 of glutathione S-transferase (GST)-MYPT1, as determined with an antibody specific for phosphorylated Thr-695. The level of Rho kinase was low in the cytoskeletal fraction and was detected primarily in the membrane and cytosolic fractions. The phosphorylation of Thr-695 by the cytoskeletal kinase(s) was not affected by Rho kinase inhibitor, Y-27632, suggesting that kinase(s) other than Rho kinase were involved. In-gel kinase assay identified a kinase at 54-59 kDa that phosphorylated the C-terminal fragment of MYPT1 (GST-MYPT1(667-1004)). Western blots detected both zipper-interacting protein kinase (ZIPK) and integrin-linked kinase (ILK) at 54-59 kDa in the cytoskeleton and membrane fractions. Cytoskeletal ZIPK and ILK were separated and partially purified by chromatography on SP-Sepharose and on MonoQ. ZIPK preferentially phosphorylated MLC20 and had low activity on MYPT1. ILK phosphorylated both MLC20 and MYPT1 and phosphorylation of MYPT1 occured on Thr-695. The above results raise the potential for regulation of MP activity in platelet cytoskeleton by ILK and suggest an alternative to the Rho-linked pathway.

    Funded by: NHLBI NIH HHS: HL 23615

    The Biochemical journal 2002;365;Pt 1;79-87

  • Role of transglutaminase II in retinoic acid-induced activation of RhoA-associated kinase-2.

    Singh US, Kunar MT, Kao YL and Baker KM

    Division of Molecular Cardiology, Cardiovascular Research Institute, The Texas A&M University System Health Science Center, College of Medicine, 1901 South 1st Street, Temple, TX 76504, USA. Usingh@medicine.tamu.edu

    Transamidation is a post-translational modification of proteins mediated by tissue transglutaminase II (TGase), a GTP-binding protein, participating in signal transduction pathways as a non-conventional G-protein. Retinoic acid (RA), which is known to have a role in cell differentiation, is a potent activator of TGASE: The activation of TGase results in increased transamidation of RhoA, which is inhibited by monodansylcadaverine (MDC; an inhibitor of transglutaminase activity) and TGaseM (a TGase mutant lacking transglutaminase activity). Transamidated RhoA functions as a constitutively active G-protein, showing increased binding to its downstream target, RhoA-associated kinase-2 (ROCK-2). Upon binding to RhoA, ROCK-2 becomes autophosphorylated and demonstrates stimulated kinase activity. The RA-stimulated interaction between RhoA and ROCK-2 is blocked by MDC and TGaseM, indicating a role for transglutaminase activity in the interaction. Biochemical effects of TGase activation, coupled with the formation of stress fibers and focal adhesion complexes, are proposed to have a significant role in cell differentiation.

    Funded by: NHLBI NIH HHS: HL60529; PHS HHS: 58439

    The EMBO journal 2001;20;10;2413-23

  • Inhibitory phosphorylation site for Rho-associated kinase on smooth muscle myosin phosphatase.

    Feng J, Ito M, Ichikawa K, Isaka N, Nishikawa M, Hartshorne DJ and Nakano T

    First Department of Internal Medicine, Mie University School of Medicine, Tsu, Mie 514-8507, Japan.

    It is clear from several studies that myosin phosphatase (MP) can be inhibited via a pathway that involves RhoA. However, the mechanism of inhibition is not established. These studies were carried out to test the hypothesis that Rho-kinase (Rho-associated kinase) via phosphorylation of the myosin phosphatase target subunit 1 (MYPT1) inhibited MP activity and to identify relevant sites of phosphorylation. Phosphorylation by Rho-kinase inhibited MP activity and this reflected a decrease in V(max). Activity of MP with different substrates also was inhibited by phosphorylation. Two major sites of phosphorylation on MYPT1 were Thr(695) and Thr(850). Various point mutations were designed for these phosphorylation sites. Following thiophosphorylation by Rho-kinase and assays of phosphatase activity it was determined that Thr(695) was responsible for inhibition. A site- and phosphorylation-specific antibody was developed for the sequence flanking Thr(695) and this recognized only phosphorylated Thr(695) in both native and recombinant MYPT1. Using this antibody it was shown that stimulation of serum-starved Swiss 3T3 cells by lysophosphatidic acid, thought to activate RhoA pathways, induced an increase in Thr(695) phosphorylation on MYPT1 and this effect was blocked by a Rho-kinase inhibitor, Y-27632. In summary, these results offer strong support for a physiological role of Rho-kinase in regulation of MP activity.

    Funded by: NHLBI NIH HHS: HL23615

    The Journal of biological chemistry 1999;274;52;37385-90

  • Localization of the gene coding for ROCK II/Rho kinase on human chromosome 2p24.

    Takahashi N, Tuiki H, Saya H and Kaibuchi K

    Central Laboratories for Key Technology, Kirin Brewery Company Ltd., 1-13-5, Fukuura, Kanazawa-ku, Yokohama, Kanagawa, 236, Japan. ntakahashi@kirin.co.jp

    Serine/threonine kinase ROCK II/Rho kinase, which is an isozyme of ROCK I, is one of the targets for the small GTPase Rho. ROCK II regulates the formation of actin stress fibers and focal adhesions, cytokinesis, smooth muscle contraction, and the activation of c-fos serum response element. To elucidate the role of ROCK II in human disease, we have cloned a 4363-bp cDNA encoding human ROCK II. Sequencing analysis has shown that human ROCK II contains 1388 amino acid residues with a calculated molecular mass of approximately 161 kDa. Fluorescence in situ hybridization analysis showed that the human ROCK II gene is located on chromosome 2p24. Radiation hybrid mapping has shown that the human ROCK II gene is located very close to the highly polymorphic marker D2S359.

    Genomics 1999;55;2;235-7

  • Prediction of the coding sequences of unidentified human genes. X. The complete sequences of 100 new cDNA clones from brain which can code for large proteins in vitro.

    Ishikawa K, Nagase T, Suyama M, Miyajima N, Tanaka A, Kotani H, Nomura N and Ohara O

    Kazusa DNA Research Institute, Kisarazu, Chiba, Japan.

    As an extension of our cDNA analysis for deducing the coding sequences of unidentified human genes, we have newly determined the sequences of 100 cDNA clones from a set of size-fractionated human brain cDNA libraries, and predicted the coding sequences of the corresponding genes, named KIAA0611 to KIAA0710. In vitro transcription-coupled translation assay was applied as the first screening to select cDNA clones which produce proteins with apparent molecular mass of 50 kDa and over. One hundred unidentified cDNA clones thus selected were then subjected to sequencing of entire inserts. The average size of the inserts and corresponding open reading frames was 4.9 kb and 2.8 kb (922 amino acid residues), respectively. Computer search of the sequences against the public databases indicated that predicted coding sequences of 87 genes were similar to those of known genes, 62% of which (54 genes) were categorized as proteins related to cell signaling/communication, cell structure/motility and nucleic acid management. The expression profiles in 10 human tissues of all the clones characterized in this study were examined by reverse transcription-coupled polymerase chain reaction and the chromosomal locations of the clones were determined by using human-rodent hybrid panels.

    DNA research : an international journal for rapid publication of reports on genes and genomes 1998;5;3;169-76

  • In mouse brain profilin I and profilin II associate with regulators of the endocytic pathway and actin assembly.

    Witke W, Podtelejnikov AV, Di Nardo A, Sutherland JD, Gurniak CB, Dotti C and Mann M

    Mouse Biology Programme, EMBL, Monterotondo/Rome, Italy. witke@embl-heidelberg.de

    Profilins are thought to be essential for regulation of actin assembly. However, the functions of profilins in mammalian tissues are not well understood. In mice profilin I is expressed ubiquitously while profilin II is expressed at high levels only in brain. In extracts from mouse brain, profilin I and profilin II can form complexes with regulators of endocytosis, synaptic vesicle recycling and actin assembly. Using mass spectrometry and database searching we characterized a number of ligands for profilin I and profilin II from mouse brain extracts including dynamin I, clathrin, synapsin, Rho-associated coiled-coil kinase, the Rac-associated protein NAP1 and a member of the NSF/sec18 family. In vivo, profilins co-localize with dynamin I and synapsin in axonal and dendritic processes. Our findings strongly suggest that in brain profilin I and profilin II complexes link the actin cytoskeleton and endocytic membrane flow, directing actin and clathrin assembly to distinct membrane domains.

    The EMBO journal 1998;17;4;967-76

  • The small GTP-binding protein Rho binds to and activates a 160 kDa Ser/Thr protein kinase homologous to myotonic dystrophy kinase.

    Ishizaki T, Maekawa M, Fujisawa K, Okawa K, Iwamatsu A, Fujita A, Watanabe N, Saito Y, Kakizuka A, Morii N and Narumiya S

    Department of Pharmacology, Kyoto University Faculty of Medicine, Japan.

    The small GTP-binding protein Rho functions as a molecular switch in the formation of focal adhesions and stress fibers, cytokinesis and transcriptional activation. The biochemical mechanism underlying these actions remains unknown. Using a ligand overlay assay, we purified a 160 kDa platelet protein that bound specifically to GTP-bound Rho. This protein, p160, underwent autophosphorylation at its serine and threonine residues and showed the kinase activity to exogenous substrates. Both activities were enhanced by the addition of GTP-bound Rho. A cDNA encoding p160 coded for a 1354 amino acid protein. This protein has a Ser/Thr kinase domain in its N-terminus, followed by a coiled-coil structure approximately 600 amino acids long, and a cysteine-rich zinc finger-like motif and a pleckstrin homology region in the C-terminus. The N-terminus region including a kinase domain and a part of coiled-coil structure showed strong homology to myotonic dystrophy kinase over 500 residues. When co-expressed with RhoA in COS cells, p160 was co-precipitated with the expressed Rho and its kinase activity was activated, indicating that p160 can associate physically and functionally with Rho both in vitro and in vivo.

    The EMBO journal 1996;15;8;1885-93

Gene lists (5)

Gene List Source Species Name Description Gene count
L00000009 G2C Homo sapiens Human PSD Human orthologues of mouse PSD adapted from Collins et al (2006) 1080
L00000016 G2C Homo sapiens Human PSP Human orthologues of mouse PSP adapted from Collins et al (2006) 1121
L00000059 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-CONSENSUS Human cortex PSD consensus 748
L00000069 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list 1461
L00000071 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-FULL Mouse cortex PSD full list (ortho) 1556
© G2C 2014. The Genes to Cognition Programme received funding from The Wellcome Trust and the EU FP7 Framework Programmes:
EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

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