cGMP-dependent protein kinase phosphorylates p21-activated kinase (Pak) 1, inhibiting Pak/Nck binding and stimulating Pak/vasodilator-stimulated phosphoprotein association.
Endothelial cells are normally non-motile and quiescent; however, endothelial cells will become permeable and invade and proliferate to form new blood vessels (angiogenesis) in response to wounding, cancer, diabetic retinopathy, age-related macular degeneration, or rheumatoid arthritis. p21-activated kinase (Pak), an effector for the Rho GTPases Rac and Cdc42, is required for angiogenesis and regulates endothelial cell permeability and motility. Although Pak is primarily activated by Rac and Cdc42, there are additional proteins that regulate Pak activity and localization, including three AGC protein kinase family members, Akt-1, PDK-1, and cAMP-dependent protein kinase. We describe phosphorylation and regulation of Pak localization by a fourth AGC kinase family member, cGMP-dependent protein kinase (PKG). Using in vitro mapping, a phosphospecific antibody, co-transfection assays, and untransfected bovine aortic endothelial cells we determined that PKG phosphorylates Pak at serine 21. Phosphorylation was accompanied by changes in proteins associated with Pak. The adaptor protein Nck was released, whereas a novel complex with vasodilator-stimulated phosphoprotein was stimulated. Furthermore Ser-21 phosphorylation of Pak appears to be important for regulation of cell morphology. In both human umbilical vein endothelial cells and HeLa cells, activation of PKG in the presence of Pak stimulated tail retraction and cell polarization. However, in cells expressing S21A mutant Pak1, PKG activation or treatment with a peptide that blocks Nck/Pak binding caused aberrant cell morphology, blocked cell retraction, and mislocalized Pak, producing uropod (tail-like) structures. These data suggest that PKG regulates Pak and that the interaction plays a role in tail retraction.
Fryer
Benjamin H
BH
Department of Pharmacology, School of Medicine, Abramson Family Cancer Research Institute, and Howard Hughes Medical Institute, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
Wang
Changhui
C
Vedantam
Srilakshmi
S
Zhou
Guo-Lei
GL
Jin
Shenghao
S
Fletcher
Linda
L
Simon
M Celeste
MC
Field
Jeffrey
J
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D019556:COS Cells / Q000378:metabolism; D002417:Cattle; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D016764:Cell Polarity; D002522:Chlorocebus aethiops; D006152:Cyclic GMP / Q000378:metabolism; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000378:metabolism; D008840:Microfilament Proteins / Q000378:metabolism*; D015513:Oncogene Proteins / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D021381:Protein Transport; D014162:Transfection; D014471:Umbilical Veins / Q000166:cytology / Q000378:metabolism; D054462:p21-Activated Kinases; D000098662:Vasodilator-Stimulated Phosphoprotein
D048868:Adaptor Proteins, Signal Transducing; D015815:Cell Adhesion Molecules; D008840:Microfilament Proteins; C078332:Nck protein; D015513:Oncogene Proteins; D010750:Phosphoproteins; D000098662:Vasodilator-Stimulated Phosphoprotein; C515903:PAK1 protein, human; D017346:Protein Serine-Threonine Kinases; D054462:p21-Activated Kinases; D017869:Cyclic GMP-Dependent Protein Kinases; D006152:Cyclic GMP