Regulation of tyrosine phosphorylation of PYK2 in vascular endothelial cells by lysophosphatidylcholine.
Lysophosphatidylcholine (LPC), a component of oxidized low-density lipoprotein, exerts various biological effects on vascular endothelial cells. However, the intracellular signaling of LPC is poorly understood. In this study, we investigated the involvement of proline-rich tyrosine kinase (PYK2) in LPC signaling in cultured bovine aortic endothelial cells by immunoprecipitation and Western blotting assays. Treatment of cells with LPC promoted a rapid increase in tyrosine phosphorylation of PYK2. LPC-stimulated PYK2 phosphorylation was inhibited by calcium chelators, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester, EGTA, protein kinase C (PKC) inhibitor, GF-109203X, or PKC depletion by phorbol esters. PYK2 phosphorylation was inhibited by treatment with cytochalasin D but with neither botulinum C3 transferase nor overexpression of a dominant negative mutant of Rho A. LPC stimulated the association of Shc with PYK2, Shc tyrosine phosphorylation, and Grb2 binding to Shc and induced Ras activation. These results provide evidence that 1) LPC tyrosine phosphorylates PYK2 by calcium- and PKC-dependent mechanisms, 2) the intact cytoskeleton is required for LPC-stimulated PYK2 phosphorylation, and 3) LPC-activated Ras via the PYK2/Shc/Grb2 signaling.
Rikitake
Y
Y
First Department of Internal Medicine, Kobe University School of Medicine, Kobe 650-0017, Japan.
Kawashima
S
S
Takahashi
T
T
Ueyama
T
T
Ishido
S
S
Inoue
N
N
Hirata
K
K
Yokoyama
M
M
D036002:ADP Ribose Transferases / Q000494:pharmacology; D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D001905:Botulinum Toxins; D002118:Calcium / Q000502:physiology; D002417:Cattle; D002478:Cells, Cultured; D015638:Cytochalasin D / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology*; D051418:Focal Adhesion Kinase 2; D051380:GRB2 Adaptor Protein; D005799:Genes, Dominant; D008244:Lysophosphatidylcholines / Q000378:metabolism* / Q000494:pharmacology; D009154:Mutation / Q000502:physiology; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000502:physiology; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D055765:Shc Signaling Adaptor Proteins; D014443:Tyrosine / Q000378:metabolism*; D018631:ras Proteins / Q000502:physiology; D020742:rhoA GTP-Binding Protein / Q000235:genetics / Q000502:physiology
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D051380:GRB2 Adaptor Protein; D008244:Lysophosphatidylcholines; D011506:Proteins; D055765:Shc Signaling Adaptor Proteins; D015638:Cytochalasin D; D014443:Tyrosine; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D011505:Protein-Tyrosine Kinases; D051418:Focal Adhesion Kinase 2; D011493:Protein Kinase C; D001905:Botulinum Toxins; D018631:ras Proteins; D020742:rhoA GTP-Binding Protein; D002118:Calcium