Differential regulation of alternatively spliced endothelial cell myosin light chain kinase isoforms by p60(Src)

The Ca2+/calmodulin-dependent endothelial cell myosin light chain kinase (M LCK) triggers actomyosin contraction essential for vascular barrier regulat ion and leukocyte diapedesis. Two high molecular weight MLCK splice variant s, EC MLCK-1 and EC MLCK-2 (210-214 kDa), in human endothelium are identica l except for a deleted single exon in MLCK-2 encoding a 69-amino acid stret ch (amino acids 436-505) that contains potentially important consensus site s for phosphorylation by p60(Src) kinase (Lazar, V., and Garcia, J. G. (199 9) Genomics 57, 256-267). We have now found that both recombinant EC MLCK s plice variants exhibit comparable enzymatic activities but a 2-fold reducti on of V-max, and a 2-fold increase in K-0.5 CaM when compared with the SM M LCK isoform, whereas K-m was similar in the three isoforms. However, only E C MLCK-1 is readily phosphorylated by purified p60(Src) in vitro, resulting in a 2- to 3-fold increase in EC MLCK-1 enzymatic activity (compared with EC MLCK-2 and SM MLCK). This increased activity of phospho-MLCK-1 was obser ved over a broad range of submaximal [Ca2+] levels with comparable EC50 [Ca 2+] for both phosphorylated and unphosphorylated EC MLCK-1. The sites of ty rosine phosphorylation catalyzed by p60(Src) are Tyr(464) and Tyr(471) with in the 69-residue stretch deleted in the MLCK-2 splice variant. These resul ts demonstrate for the first time that p60(Src)-mediated tyrosine phosphory lation represents an important mechanism for splice variant-specific regula tion of nonmuscle MLCK and vascular cell function.