PYK2 as a mediator of endothelin-1/G alpha 11 signaling to GLUT4 glucose transporters.
Endothelin-1 (ET-1) signaling through G alpha(q/11) stimulates translocation of intracellular GLUT4 glucose transporters to the plasma membrane of 3T3-L1 adipocytes by an unknown mechanism that requires protein tyrosine phosphorylation and ADP-ribosylation factor 6 (ARF6) but is independent of phosphatidylinositol 3 (PI3)-kinase. In contrast, insulin action on this process requires PI3-kinase but not ARF6. Here we report the identification of two proteins selectively tyrosine-phosphorylated in response to ET-1 but not insulin: the Ca(2+)-activated tyrosine kinase PYK2 and its physiological substrate, the adhesion scaffold protein paxillin. Endogenous paxillin as well as expressed Myc-tagged PYK2 or a Myc-tagged kinase-deficient PYK2 protein were acutely directed to F-actin-rich adhesion sites from the adipocyte cytoplasm in response to ET-1 but not insulin. CADTK-related non-kinase (CRNK) is a dominant negative form of PYK2 containing the C-terminal portion of the protein, which binds paxillin but lacks the PYK2 autophosphorylation site (Tyr(402)). CRNK expression in 3T3-L1 adipocytes inhibited ET-1-mediated F-actin polymerization and translocation of Myc-tagged GLUT4-enhanced green fluorescent protein (EGFP) to the plasma membrane without disrupting insulin action on these processes. These data reveal the tyrosine kinase PYK2 as a required signaling element in the regulation of GLUT4 recycling in 3T3-L1 adipocytes by ET-1, whereas insulin signaling is directed through a different pathway.
Park
J G
JG
Program in Molecular Medicine and Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.
Bose
A
A
Leszyk
J
J
Czech
M P
MP
D016475:3T3 Cells; D000089982:ADP-Ribosylation Factor 6; D000818:Animals; D019332:Endothelin-1 / Q000378:metabolism*; D051418:Focal Adhesion Kinase 2; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D051275:Glucose Transporter Type 4; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism*; D051379:Mice; D008856:Microscopy, Fluorescence; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D009124:Muscle Proteins; D021381:Protein Transport; D011505:Protein-Tyrosine Kinases / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology*
D000089982:ADP-Ribosylation Factor 6; D019332:Endothelin-1; D051275:Glucose Transporter Type 4; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; C495693:Slc2a4 protein, mouse; D011505:Protein-Tyrosine Kinases; D051418:Focal Adhesion Kinase 2; C496053:Ptk2b protein, mouse; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins; C000715727:Arf6 protein, mouse