Differential regulation of alternatively spliced endothelial cell myosin light chain kinase isoforms by p60(Src).
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 (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 [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.
Birukov
K G
KG
Division of Pulmonary and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21224, USA.
Csortos
C
C
Marzilli
L
L
Dudek
S
S
Ma
S F
SF
Bresnick
A R
AR
Verin
A D
AD
Cotter
R J
RJ
Garcia
J G
JG
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D004730:Endothelium, Vascular / Q000201:enzymology*; D004789:Enzyme Activation; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism*; D010766:Phosphorylation; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000502:physiology*; D011817:Rabbits; D014443:Tyrosine / Q000378:metabolism; D018909:src Homology Domains
D007527:Isoenzymes; D014443:Tyrosine; D016392:Proto-Oncogene Proteins pp60(c-src); D009219:Myosin-Light-Chain Kinase