Identification and characterization of a novel family of serine/threonine kinases containing two N-terminal LIM motifs.
We previously isolated human cDNA coding for LIMK1 (LIM motif-containing protein kinase-1), a putative protein kinase containing two LIM motifs at the N terminus and an unusual protein kinase domain at the C terminus. In the present study, we isolated human cDNA encoding LIMK2, a second member of a LIMK family, with a domain structure similar to LIMK1 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 LIMK1 and LIMK2 mRNAs in human tissues differ significantly. Chromosomal localization of human LIMK1 and LIMK2 genes was assigned to 7q11.23 and 22q12, respectively, by fluorescence in situ hybridization. The Myc epitope-tagged LIMK1 and LIMK2 proteins transiently expressed in COS cells exhibited serine/threonine-specific kinase activity toward myelin basic protein and histone in in vitro kinase assay. Immunofluorescence and subcellular fractionation analysis revealed that Myc-tagged LIMK1 and LIMK2 were localized mainly in the cytoplasm. The "native" LIMK1 protein endogenously expressed in A431 epidermoid carcinoma cells also exhibited serine/threonine kinase activity. The specific activity of native LIMK1 from A431 cells was apparently much higher than that of "recombinant" LIMK1 ectopically expressed in COS cells, hence, it is likely that there is a mechanism, by which native LIMK1 is activated. A 140-kDa tyrosine-phosphorylated protein (pp140) was co-immunoprecipitated with native LIMK1 form A431 cell lysates; therefore, pp140 may be a LIMK1-associated protein involved in the regulation of LIMK1 function.
Okano
I
I
Department of Biology, Faculty of Science, Kyushu University, Fukuoka, Japan.
Hiraoka
J
J
Otera
H
H
Nunoue
K
K
Ohashi
K
K
Iwashita
S
S
Hirai
M
M
Mizuno
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002874:Chromosome Mapping; D002892:Chromosomes, Human, Pair 22; D002897:Chromosomes, Human, Pair 7; D018076:DNA, Complementary; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D054461:Lim Kinases; D008969:Molecular Sequence Data; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions / Q000201:enzymology
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D011494:Protein Kinases; C515892:LIMK1 protein, human; C102895:LIMK2 protein, human; D054461:Lim Kinases; D017346:Protein Serine-Threonine Kinases