PKD1/PKCmu promotes alphavbeta3 integrin recycling and delivery to nascent focal adhesions.
To identify kinases that regulate integrin recycling, we have immunoprecipitated alphavbeta3 integrin from NIH 3T3 fibroblasts in the presence and absence of primaquine (a drug that inhibits receptor recycling and leads to accumulation of integrins in endosomes) and screened for co-precipitating kinases. Primaquine strongly promoted association of alphavbeta3 integrin with PKD1, and fluorescence microscopy indicated that integrin and PKD1 associate at a vesicular compartment that is downstream of a Rab4-dependent transport step. PKD1 association was mediated by the C-terminal region of the beta3 integrin cytodomain, and mutants of beta3 that were unable to recruit PKD1 did not recycle in a PDGF-dependent fashion. Furthermore, suppression of endogenous PKD1 levels by RNAi, or overexpression of catalytically inactive PKD1 inhibited PDGF-dependent recycling of alphavbeta3 from early endosomes to the plasma membrane and blocked recruitment of alphavbeta3 to newly formed focal adhesions during cell spreading. These data indicate that PKD1 influences cell migration by directing vesicular transport of the alphavbeta3 integrin heterodimer.
Woods
Alison J
AJ
Department of Biochemistry, University of Leicester, Leicester, UK.
White
Dominic P
DP
Caswell
Patrick T
PT
Norman
Jim C
JC
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D015153:Blotting, Western; D002465:Cell Movement; D002478:Cells, Cultured; D011992:Endosomes / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005347:Fibroblasts / Q000737:chemistry / Q000378:metabolism; D022001:Focal Adhesions / Q000187:drug effects*; D005982:Glutathione Transferase / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D039302:Integrin alphaVbeta3 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008280:Magnetics; D051379:Mice; D018613:Microscopy, Confocal; D009154:Mutation; D041681:NIH 3T3 Cells; D010982:Platelet-Derived Growth Factor / Q000378:metabolism / Q000494:pharmacology; D011233:Precipitin Tests; D011319:Primaquine / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000378:metabolism*; D034622:RNA Interference; D019030:Receptors, Vitronectin / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D050396:TRPP Cation Channels; D014162:Transfection
D000911:Antibodies, Monoclonal; D005982:Glutathione Transferase; D049452:Green Fluorescent Proteins; D039302:Integrin alphaVbeta3; D010982:Platelet-Derived Growth Factor; D011319:Primaquine; D011493:Protein Kinase C; D011506:Proteins; D019030:Receptors, Vitronectin; D011993:Recombinant Fusion Proteins; D050396:TRPP Cation Channels; C089212:protein kinase D