Differential Regulation of Alternatively Spliced Endothelial Cell Myosin Light Chain Kinase Isoforms by p60Src

Konstantin G. Birukov, Csilla Csortos, Lisa Marzilli, Steven Dudek, Shwu-Fan Ma, Anne R. Bresnick, Alexander D. Verin, Robert J. Cotter, Joe G. N. Garcia
2000 Journal of Biological Chemistry  
The Ca 2؉ /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
more » ... , 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 [Ca 2؉ ] levels with comparable EC 50 [Ca 2؉ ] for both phosphorylated and unphosphorylated EC MLCK-1. The sites of tyrosine phosphorylation catalyzed by p60 Src are Tyr 464 and Tyr 471 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 nonmuscle MLCK and vascular cell function.
doi:10.1074/jbc.m005270200 pmid:11113114 fatcat:wfoip3cos5fplorhgbfgtxb7wa