14-691 Sigma-AldrichMRCKα Protein, active, 10 µg

The myotonic dystrophy kinase-related kinases RhoA binding kinase and myotonic dystrophy kinase-related Cdc42 binding kinase (MRCK) are effectors of RhoA and Cdc42, respectively, for actin reorganization. Using substrate screening in various tissues, we uncovered two major substrates, p130 and p85, for MRCKalpha-kinase. p130 is identified as myosin binding subunit p130, whereas p85 is a novel related protein. p85 contains N-terminal ankyrin repeats, an alpha-helical C terminus with leucine repeats, and a centrally located conserved motif with the MRCKalpha-kinase phosphorylation site. Like MBS130, p85 is specifically associated with protein phosphatase 1delta (PP1delta), and this requires the N terminus, including the ankyrin repeats. This association is required for the regulation of both the catalytic activities and the assembly of actin cytoskeleton. The N terminus, in association with PP1delta, is essential for actin depolymerization, whereas the C terminus antagonizes this action. The C-terminal effects consist of two independent events that involved both the conserved phosphorylation inhibitory motif and the alpha-helical leucine repeats. The former was able to interact with PP1delta only in the phosphorylated state and result in inactivation of PP1delta activity. This provides further evidence that phosphorylation of a myosin binding subunit protein by specific kinases confers conformational changes in a highly conserved region that plays an essential role in the regulation of its catalytic subunit activities.

The Rho GTPases play distinctive roles in cytoskeletal reorganization associated with growth and differentiation. The Cdc42/Rac-binding p21-activated kinase (PAK) and Rho-binding kinase (ROK) act as morphological effectors for these GTPases. We have isolated two related novel brain kinases whose p21-binding domains resemble that of PAK whereas the kinase domains resemble that of myotonic dystrophy kinase-related ROK. These approximately 190-kDa myotonic dystrophy kinase-related Cdc42-binding kinases (MRCKs) preferentially phosphorylate nonmuscle myosin light chain at serine 19, which is known to be crucial for activating actin-myosin contractility. The p21-binding domain binds GTP-Cdc42 but not GDP-Cdc42. The multidomain structure includes a cysteine-rich motif resembling those of protein kinase C and n-chimaerin and a putative pleckstrin homology domain. MRCK alpha and Cdc42V12 colocalize, particularly at the cell periphery in transfected HeLa cells. Microinjection of plasmid encoding MRCK alpha resulted in actin and myosin reorganization. Expression of kinase-dead MRCK alpha blocked Cdc42V12-dependent formation of focal complexes and peripheral microspikes. This was not due to possible sequestration of the p21, as a kinase-dead MRCK alpha mutant defective in Cdc42 binding was an equally effective blocker. Coinjection of MRCK alpha plasmid with Cdc42 plasmid, at concentrations where Cdc42 plasmid by itself elicited no effect, led to the formation of the peripheral structures associated with a Cdc42-induced morphological phenotype. These Cdc42-type effects were not promoted upon coinjection with plasmids of kinase-dead or Cdc42-binding-deficient MRCK alpha mutants. These results suggest that MRCK alpha may act as a downstream effector of Cdc42 in cytoskeletal reorganization.

We have cloned a novel serine/threonine protein kinase (PK428) which is highly related (65%) within the kinase domain to the myotonic dystrophy protein kinase (DM-PK), as well as the cyclic AMP-dependent protein kinase (33%). Northern blots demonstrate that PK428 mRNA is distributed widely among tissues and is expressed at the highest levels in pancreas, heart, and skeletal muscle, with lower levels in liver and lung. Two PK428 mRNAs 10 and 3.8 kilobase pairs in size are seen in a number of cell lines, including hematopoietic and breast cancer cells. An antibody generated to a glutathione S-transferase-PK428 fusion protein detects a 65-kDa protein in these cell lines, and a similarly sized protein when the cloned cDNA is transiently expressed in Cos 7 cells. Immunoprecipitation of the transiently expressed PK428 protein and incubation with [gamma-32P]ATP demonstrate that it is capable of autophosphorylation. In addition, immunoprecipitates of the PK428 protein kinase also phosphorylated histone H1 and a peptide encoding a cyclic AMP-dependent protein kinase substrate. The gene corresponding to the 3.8-kb PK428 mRNA, and its corresponding 65-kDa protein, was isolated by polymerase chain reaction screening of a P1 phage human genomic library. Using this P1 phage clone as a probe, the PK428 gene was located on 1q41-42, a possible location for a human senescence gene, a gene associated with Rippling muscle disease, as well as a region associated with genetically acquired mental retardation.