CLONING AND BIOCHEMICAL-CHARACTERIZATION OF LIMK-2, A PROTEIN-KINASE CONTAINING 2 LIM DOMAINS

We have isolated human and rat clones of the LIM motif-containing prot ein kinase, termed LIMK-2, LIMK-2 is related to the neuronally express ed LIM-kinase, whose hemizygous deletion appears to result in cognitiv e impairment in patients with Williams syndrome. The hallmark of this protein family is the presence of I or 2 N-terminal LIM motifs and an atypical C-terminal protein kinase domain. LIMK-2 mRNA was detected by Northern blot analysis is human tissues, most abundantly in placenta, lung, liver, and pancreas, and also in a variety of cell lines includ ing neuronal, glioblastoma, and mammary carcinoma lines. The LIMK-2 tr anscript was also induced upon neuroectodermal differentiation of mous e P19 embryonal carcinoma cells. A 65 kDa recombinant LIMK-2 protein w as identified in 293 cells stably transfected with a LIMK-2 expression vector. An in vitro kinase assay demonstrates LIMK-2 is autophosphory lated and exhibits serine/threonine kinase activity towards the exogen ous substrate MBP. The endogenous 65 kDa LIMK-2 protein was detected i n a variety of cell lines, and coprecipitates with a 140 kDa tyrosine phosphorylated protein, but was not itself tyrosine phosphorylated. At the subcellular level, LIMK-2 is localized in both the nucleus and in a Triton X-100 soluble fraction.