Intermolecular and intramolecular interactions regulate catalytic activity of myotonic dystrophy kinase-related Cdc42-binding kinase alpha.
Myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK) is a Cdc42-binding serine/threonine kinase with multiple functional domains. We had previously shown MRCKalpha to be implicated in Cdc42-mediated peripheral actin formation and neurite outgrowth in HeLa and PC12 cells, respectively. Here we demonstrate that native MRCK exists in high-molecular-weight complexes. We further show that the three independent coiled-coil (CC) domains and the N-terminal region preceding the kinase domain are responsible for intermolecular interactions leading to MRCKalpha multimerization. N terminus-mediated dimerization and consequent transautophosphorylation are critical processes regulating MRCKalpha catalytic activities. A region containing the two distal CC domains (CC2 and CC3; residues 658 to 930) was found to interact intramolecularly with the kinase domain and negatively regulates its activity. Its deletion also resulted in an active kinase, confirming a negative autoregulatory role. We provide evidence that the N terminus-mediated dimerization and activation of MRCK and the negative autoregulatory kinase-distal CC interaction are two mutually exclusive events that tightly regulate the catalytic state of the kinase. Disruption of this interaction by a mutant kinase domain resulted in increased kinase activity. MRCK kinase activity was also elevated when cells were treated with phorbol ester, which can interact directly with a cysteine-rich domain next to the distal CC domain. We therefore suggest that binding of phorbol ester to MRCK releases its autoinhibition, allowing N-terminal dimerization and subsequent kinase activation.
Glaxo-IMCB Group, Institute of Molecular & Cell Biology, Singapore 117609, Singapore.
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D000595:Amino Acid Sequence; D000818:Animals; D017931:DNA Primers / Q000235:genetics; D019281:Dimerization; D006367:HeLa Cells; D006801:Humans; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D065636:Myotonin-Protein Kinase; D016716:PC12 Cells; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D051381:Rats; D017386:Sequence Homology, Amino Acid; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism*
C585517:DMPK protein, human; D017931:DNA Primers; D046911:Macromolecular Substances; D065636:Myotonin-Protein Kinase; D017346:Protein Serine-Threonine Kinases; D020764:cdc42 GTP-Binding Protein