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

Research output: Contribution to journalArticle

Abstract

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 p60Src 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 Vmax, and a 2-fold increase in K0.5 CaM when compared with the SM MLCK isoform, whereas Km was similar in the three isoforms. However, only EC MLCK-1 is readily phosphorylated by purified p60Src 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 p60Src 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 p60Src-mediated tyrosine phosphorylation represents an important mechanism for splice variant-specific regulation of non-muscle MLCK and vascular cell function.

Original languageEnglish (US)
Pages (from-to)8567-8573
Number of pages7
JournalJournal of Biological Chemistry
Volume276
Issue number11
DOIs
StatePublished - Mar 16 2001

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Birukov, K. G., Csortos, C., Marzilli, L., Dudek, S., Ma, S. F., Bresnick, A. R., Verin, A. D., Cotter, R. J., & Garcia, J. G. N. (2001). Differential Regulation of Alternatively Spliced Endothelial Cell Myosin Light Chain Kinase Isoforms by p60Src. Journal of Biological Chemistry, 276(11), 8567-8573. https://doi.org/10.1074/jbc.M005270200