IDENTIFICATION AND CHARACTERIZATION OF A NOVEL FAMILY OF SERINE THREONINE KINASES CONTAINING 2 N-TERMINAL LIM MOTIFS

We previously isolated human cDNA coding for LIMK1 (LIM motif containi ng protein kinase-1), a putative protein kinase containing two LIM mot ifs at the N terminus and an unusual protein kinase domain at the C te rminus, In the present study, we isolated human cDNA encoding LIMK2, a second member of a LIMK family, with a domain structure similar to LI MK1 and 50% overall amino acid identity with LIMK1. The protein kinase domains of LIMK1 and LIMK2 are unique in that they contain an unusual sequence motif Asp Leu-Asn-Ser-His-Asn in subdomain VIB and a highly basic insert between subdomains VII and VIII, Expression patterns of L IMK1 and LIMK2 mRNAs in human tissues differ significantly. Chromosoma l localization of human LIMK1 and LIMK2 genes was assigned to 7q11.23 and 22q12, respectively, by fluorescence in situ hybridization, The My c epitope-tagged LIMK1 and LIMK2 proteins transiently expressed in COS cells exhibited serine/threonine-specific kinase activity toward myel in basic protein and histone in in vitro kinase assay. Immunofluoresce nce and subcellular fractionation analysis revealed that Myc-tagged LI MK1 and LIMK2 were localized mainly in the cytoplasm, The ''native'' L IMK1 protein endogenously expressed in A431 epidermoid carcinoma cells also exhibited serine/threonine kinase activity. The specific activit y of native LIMK1 from A431 cells was apparently much higher than that of ''recombinant'' LIMK1 ectopically expressed in COS cells, hence, i t is likely that there is a mechanism, by which native LIMK1 is activa ted, A 140-kDa tyrosine phosphorylated protein (pp140) was co-immunopr ecipitated with native LIMK1 from A431 cell lysates; therefore, pp140 may be a LIMK1-associated protein involved in the regulation of LIMK1 function.