genghis khan

EVOLUTIONARY HOMOLOGS

Gek exhibits strong sequence similarity to human myotonic dystrophy protein kinase (DMPK). Gek and DMPK share 63% amino acid sequence identity within the 271-aa catalytic core, the sequence similarity between these two proteins extending beyond the catalytic domin in both directions. DMPK, however, is much smaller than Gek. Interestingly, members of a recently identified class of Rho binding kinases, which may function as effectors of the small GTPase Rho, have been found to be similar to Gek in their domain structures. The kinase domain of the Rho-binding kinase is also similar to DMPK, although DMPK is more similar to Drosophila Gek than to mammalian Rho-kinase. The phorbol ester binding domain of protein kinase C bears a stronger similarity to the Cys-rich domain of Gek than to that of Rho-binding kinase (Luo, 1997).

Using positional cloning strategies, a CTG triplet repeat has been identified that undergoes expansion in myotonic dystrophy patients. This sequence is highly variable in the normal population. PCR analysis of the interval containing this repeat indicates that unaffected individuals have between 5 and 27 copies. Myotonic dystrophy patients who are minimally affected have at least 50 repeats, while more severely affected patients have expansion of the repeat containing segment up to several kilobase pairs. The CTG repeat is transcribed and is located in the 3' untranslated region of an mRNA that is expressed in tissues affected by myotonic dystrophy. This mRNA encodes a polypeptide that is a member of the protein kinase family (Brook, 1992).

Two genes have been identified that are associated with the hypodermal cell shape changes that occur during elongation of the Caenorhabditis elegans embryo. The first gene, called let-502, encodes a protein with high similarity to Rho-binding Ser/Thr kinases and to human myotonic dystrophy kinase (DM-kinase). Strong mutations in let-502 block embryonic elongation; let-502 reporter constructs are expressed in hypodermal cells at the elongation stage of development. The second gene, mel-11, was identified by mutations that act as extragenic suppressors of let-502. mel-11 encodes a protein similar to the 110- to 133-kD regulatory subunits of vertebrate smooth muscle myosin-associated phosphatase (PP-1M). It is suggested that the LET-502 kinase and the MEL-11 phosphatase subunit act in a pathway linking a signal generated by the small GTP-binding protein Rho to a myosin-based hypodermal contractile system that drives embryonic elongation. LET-502 may directly regulate the activity of the MEL-11 containing phosphatase complex: the similarity between LET-502 and DM-kinase suggests a similar function for DM-kinase (Wissmann, 1997).

Myotonic dystrophy (DM) is associated with expansion of CTG repeats in the 3'-untranslated region of the myotonin protein kinase (DMPK) gene. The molecular mechanism whereby expansion of the (CUG)n repeats in the 3'-untranslated region of DMPK gene induces DM is unknown. A protein has been isolated with specific binding to CUG repeat sequences (CUG-BP/hNab50) that possibly plays a role in mRNA processing and/or transport. The phosphorylation status and intracellular distribution of the RNA CUG-binding protein (a potential homolog of Drosophila Arrest, also known as Bruno), identical to hNab50 protein (CUG-BP/hNab50), are altered in homozygous DM patients. CUG-BP/hNab50 is a substrate for DMPK both in vivo and in vitro. Data from two biological systems with reduced levels of DMPK ( homozygous DM patients and DMPK knockout mice) shows that DMPK regulates both phosphorylation and intracellular localization of the CUG-BP/hNab50 protein. Decreased levels of DMPK observed in both the DM patients and DMPK knockout mice led to the elevation of the hypophosphorylated form of CUG-BP/hNab50. Nuclear concentration of the hypophosphorylated CUG-BP/hNab50 isoform is increased in DMPK knockout mice and in homozygous DM patients. DMPK also interacts with and phosphorylates CUG-BP/hNab50 protein in vitro. DMPK-mediated phosphorylation of CUG-BP/hNab50 results in the dramatic reduction of CUG-BP2, the hypophosphorylated isoform, accumulation of which is observed in the nuclei of DMPK knockout mice. These data suggest a feedback mechanism whereby decreased levels of DMPK could alter the phosphorylation status of CUG-BP/hNab50, thus facilitating nuclear localization of CUG-BP/hNab50. These results suggest that DM pathophysiology could be, in part, a result of sequestration of CUG-BP/hNab50 and, in part, of lowered DMPK levels, which, in turn, affect processing and transport of specific subclass of mRNAs (Roberts, 1997).

The small GTP-binding protein Rho functions as a molecular switch in the formation of focal adhesions and stress fibers and in the activation of cytokinesis and transcription. The biochemical mechanism underlying these actions remains unknown. Using a ligand overlay assay, a 160 kDa platelet protein has been purified that binds specifically to GTP-bound Rho. This protein, p160, undergoes autophosphorylation at its serine and threonine residues and demonstrates kinase activity to exogenous substrates. Both activities are enhanced by the addition of GTP-bound Rho. A cDNA encoding the gene p160 codes 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 a coiled-coil structure shows strong homology to myotonic dystrophy kinase over 500 residues. When co-expressed with RhoA in COS cells, p160 is co-precipitated with the expressed Rho and its kinase activity is activated, indicating that p160 can associate physically and functionally with Rho both in vitro and in vivo (Ishizaki, 1996).

A rat cDNA encoding a 150-kDa protein resembling DMPK has been isolated that specifically binds RhoA in its GTP form and contains an N-terminal serine/threonine kinase domain highly related to the human myotonic dystrophy kinase and a cysteine-rich domain toward the C terminus. The RhoA binding domain is unrelated to other p21 binding domains. Antibody raised against the kinase domain of the predicted protein, termed ROK alpha (for ROK alpha, RhoA-binding kinase), recognizes a ubiquitous 150-kDa protein. The brain p150 purified by affinity chromatography with RhoA exhibits serine/threonine kinase activity. In cultured cells, immunoreactive p150 is recruited to membranes upon transfection with dominant positive RhoAV14 mutant and is localized with actin microfilaments at the cell periphery. These results are consistent with a role for the kinase ROK alpha as an effector for RhoA (Leung, 1995).

A Rho-interacting protein with a molecular mass of approximately 164 kDa (p164) from bovine brain, resembling DMPK has been characterized. This protein binds to GTPgammaS (a non-hydrolyzable GTP analog).RhoA but not to GDP.RhoA or GTPgammaS.RhoA, which has a mutation in the effector domain (RhoAA37). p164 has a kinase activity that was specifically stimulated by GTPgammaS.RhoA. The cDNA encoding p164 was obtained on the basis of its partial amino acid sequences and was named Rho-associated kinase (Rho-kinase). Rho-kinase has a catalytic domain in the N-terminal portion, a coiled coil domain in the middle portion and a zinc finger-like motif in the C-terminal portion. The catalytic domain shares 72% sequence homology with that of myotonic dystrophy kinase, and the coiled coil domain contains a Rho-interacting interface. When COS7 cells were cotransfected with Rho-kinase and activated RhoA, some Rho-kinase was recruited to membranes. Thus it is likely that Rho-kinase is a putative target serine/threonine kinase for Rho and serves as a mediator of the Rho-dependent signaling pathway (Matsui, 1996).

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. Two related novel brain kinases have been isolated 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 that 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 results in actin and myosin reorganization. Expression of kinase-dead MRCK alpha blocks Cdc42V12-dependent formation of focal complexes and peripheral microspikes. This was not due to possible sequestration of the p21, since a kinase-dead MRCK alpha mutant, defective in Cdc42 binding, is an equally effective blocker. Coinjection of MRCK alpha plasmid with Cdc42 plasmid, at concentrations where Cdc42 plasmid by itself elicits no effect, leads to the formation of the peripheral structures associated with a Cdc42-induced morphological phenotype. These Cdc42-type effects are 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 (Leung, 1998).

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