Characterization of RhoA-binding kinase ROKalpha implication of the pleckstrin homology domain in ROKalpha function using region-specific antibodies.
Rho-binding kinase alpha (ROKalpha) is a serine/threonine kinase with multiple functional domains involved in actomyosin assembly. It has previously been documented that the C terminus part of ROKalpha interacts with the N-terminal kinase domain and thereby regulates its catalytic activity. Here we used antibodies against different domains of ROKalpha and were able to reveal some structural aspects that are essential for the specific functions of ROKalpha. Antibodies against the kinase domain revealed that this part of the protein is highly complex and inaccessible. Further experiments confirmed that this domain could undergo inter- and intramolecular interactions in a complex manner, which regulates the kinase catalytic activity. Other antibodies that raised against the coiled-coil domain, Rho binding domain, and the pleckstrin homology (PH) domain were all effective in recognizing the native proteins in an immunoprecipitation assay. Only the anti-Rho binding domain antibodies could activate the kinase independent of RhoA. The PH antibodies had no apparent effects on the catalytic activity but were effective in blocking actomyosin assembly and cell contractility. Likewise, mutations of the PH domains can abrogate its dominant negative effects on actin morphology. The subsequent disruption of endogenous ROK localization to the actomyosin network by overexpressing the PH domain is supportive of a role of the PH domain of ROK in targeting the kinase to these structures.
Chen
Xiang-qun
XQ
Glaxo-IMCB Group, Institute of Molecular and Cell Biology, 30 Medical Dr., Singapore 117609, Singapore.
Tan
Ivan
I
Ng
Chong Han
CH
Hall
Christine
C
Lim
Louis
L
Leung
Thomas
T
D000818:Animals; D000918:Antibody Specificity; D001483:Base Sequence; D001798:Blood Proteins / Q000737:chemistry*; D019556:COS Cells; D002384:Catalysis; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D047908:Intracellular Signaling Peptides and Proteins; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009218:Myosins / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry*; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D011994:Recombinant Proteins; D054460:rho-Associated Kinases
D001798:Blood Proteins; D017931:DNA Primers; D018076:DNA, Complementary; D047908:Intracellular Signaling Peptides and Proteins; D010750:Phosphoproteins; D011994:Recombinant Proteins; C039077:platelet protein P47; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D009218:Myosins