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 (MLCK) triggers actomyosin contraction essential for vascular barrier regulation and leukocyte diapedesis. Two high molecular weight MLCK splice variants, EC MLCK-1 and EC MLCK-2 (210-214 kDa), in human endothelium are identical except for a deleted single exon in MLCK-2 encoding a 69-amino acid stretch (amino acids 436-505) that contains potentially important consensus sites for phosphorylation by p60^Src kinase (Lazar, V., and Garcia, J. G. (1999) Genomics 57, 256-267). We have now found that both recombinant EC MLCK splice variants exhibit comparable enzymatic activities but a 2-fold reduction of V_max, and a 2-fold increase in K_{0.5 CaM} when compared with the SM MLCK isoform, whereas K_m was similar in the three isoforms. However, only EC 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 observed over a broad range of submaximal [Ca²⁺] levels with comparable EC₅₀ [Ca²⁺] for both phosphorylated and unphosphorylated EC MLCK-1. The sites of tyrosine phosphorylation catalyzed by p60^Src are Tyr ⁴⁶⁴ and Tyr⁴⁷¹ within the 69-residue stretch deleted in the MLCK-2 splice variant. These results demonstrate for the first time that p60^Src-mediated tyrosine phosphorylation represents an important mechanism for splice variant-specific regulation of non-muscle MLCK and vascular cell function.