Identification and Characterization of a Novel Family of Serine/Threonine Kinases Containing Two N-terminal LIM Motifs

Abstract

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-SerHis-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 from A431 cell lysates; therefore, pp140 may be a LIMK1-associated protein involved in the regulation of LIMK1 function.Protein kinases play a central role in the intracellular signal transduction systems involved in cell proliferation, differentiation, metabolism, and other responses to external stimuli. Numerous protein kinases have been identified, and their cellular functions have been studied extensively (1, 2). However, our understanding of the mechanisms of cellular signaling systems is still limited. Further searches for a novel class of protein kinases and elucidation of their roles in various systems are important in unravelling the various mechanisms of signaling underlying diverse cell activities.We previously identified the human cDNA encoding a novel putative protein kinase, termed LIM-kinase (LIMK) 1 (3). LIMK has characteristic structural features in that it is composed of two LIM/double zinc finger motifs at the N terminus and an unusual protein kinase domain at the C terminus. The cDNA sequences encoding chicken and mouse LIMK, with similar structural characteristics, were subsequently reported (4, 5). The LIM motif, named after the capitals of three homeodomain-containing proteins, Lin-11, Isl-1, and Mec-3, was defined as a paired zinc finger motif composed of 50 -60 amino acid residues with a conserved placement of cysteine and histidine (and aspartic acid) residues, (CX 2 CX 16 -19 HX 2 C)...

Inhibition of activated Ras-induced neuronal differentiation of PC12 cells by the LIM domain of LIM-kinase 1

“…In addition to shifts in gene expression, MAPK activation also induces dynamic cytoskeletal rearrangements leading to morphological changes, such as an increase of cell mass and neurite outgrowth, in PC12 cells. Since LIMKs are mainly localized in the cytoplasm (see Figure 1d and Okano et al, 1995), they a ect PC12 cell di erentiation probably by interfering with the events related to cytoskeletal reorganization, although the possibility that LIMKs perturb the nuclear events controlling gene expression cannot be excluded at present.…”

“…We recently identi®ed human, rat and chicken cDNAs encoding a novel class of protein kinases, termed LIMkinases (LIMKs), composed of LIMK1 and LIMK2 Ohashi et al, 1994;Okano et al, 1995;Nunoue et al, 1995). The cDNA corresponding to mouse LIMK1 was identi®ed by other groups (Bernard et al, 1994;Cheng and Robertson, 1995;ProÈ schel et al, 1995).…”

The cofilin pathway in breast cancer invasion and metastasis

“…Cofilin is then regulated by four independent processes. First, the phosphorylation of cofilin on serine 3 by LIM kinase 1 (LIMK1) and its related kinases (LIMK2, the skeletal muscle-specific kinases Nik related kinase (NRK, also known as NESK) and testicular protein kinase 1 (TESK1) and TESK2) regulates cofilin by inhibiting its actin-binding activity [19][20][21][22] ; second, the dephosphorylation of serine 3 of cofilin by phosphatase types 1, 2A and 2B, slingshot (SSH) and chronophin phosphatases results in the activation of actin binding by cofilin [23][24][25][26] . Third, actin binding by cofilin is inhibited by binding to phosphatidylinositol-4,5-bisphosphate (PIP2) 27,28 , and cofilin activity is dependent on phospho lipase Cγ (PLCγ)-mediated hydrolysis of PIP2 (REFS 29,30); and fourth, changes in pH over the physiological range (6.8-7.4) as mediated by the Na-H exchanger protein can activate the severing activity of cofilin when it is in the dephosphorylated state [31][32][33][34] …”

Comprehensive expression analysis of a rat depression model

“…Additional reports that depressive patients frequently manifest subcortical hyperintensity near frontal white matter ('myelin pallor' on histological examination) 39 suggest that LIMK1 may be involved in an as yet undetermined intercellular signaling pathway disrupted in depression, as LIMK1 is known to phosphorylate myelin basic proteins. 40 Our criteria for selecting 'altered' transcripts in LH compared to control animals may have been conservative and inadvertently excluded many potential candidates. Decreased levels of brain-derived neurotrophic factor (BDNF) recoverable by antidepressant treatment have been reported in patients with depression.…”