Phosphatidylinositol 3-kinase and Src family kinases are required for phosphorylation and membrane recruitment of Dok-1 in c-Kit signaling.
Dok-1 is an adaptor protein that is a substrate for Bcr-Abl and other tyrosine protein kinases. The presence of pleckstrin homology and phosphotyrosine binding domains as well as multiple tyrosine phosphorylation sites suggests that Dok-1 is involved in protein-protein and/or protein-lipid interactions. Here we show that stimulation of Mo7 hematopoietic cells with c-Kit ligand (KL) induces phosphatidylinositol (PI) 3-kinase-dependent tyrosine phosphorylation and membrane recruitment of Dok-1. Addition of the K-Ras membrane-targeting motif to Dok-1 generated a constitutively membrane-bound Dok-1 protein whose tyrosine phosphorylation was independent of PI 3-kinase. Membrane localization of Dok-1 was required for its ability to function as a negative regulator of cell proliferation. Additional experiments revealed that Dok-1 associated with the juxtamembrane region and C-terminal tail of c-Kit. Lyn promoted phosphorylation of c-Kit and association of c-Kit and Dok-1. Both Lyn and Tec were capable of phosphorylating Dok-1. However, the use of primary bone marrow mast cells from normal and Lyn-deficient mice demonstrated that Lyn is required for KL-dependent Dok-1 tyrosine phosphorylation. Taken together, these data indicate that activation of PI 3-kinase by KL promotes binding of the Dok pleckstrin homology domain and Dok-1 recruitment to the plasma membrane where Dok-1 is phosphorylated by Src and/or Tec family kinases.
Liang
Xiquan
X
Cell Biology Program and the Molecular Pharmacology and Therapeutics Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
Wisniewski
David
D
Strife
Annabel
A
Shivakrupa
Clarkson
Bayard
B
Resh
Marilyn D
MD
D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D019556:COS Cells; D002455:Cell Division; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D050356:Lipid Metabolism; D051379:Mice; D019869:Phosphatidylinositol 3-Kinases / Q000737:chemistry / Q000502:physiology*; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D019009:Proto-Oncogene Proteins c-kit / Q000737:chemistry / Q000378:metabolism*; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D013347:Subcellular Fractions; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism; D000077191:Wortmannin; D019061:src-Family Kinases / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*
D000730:Androstadienes; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; C489560:DOK1 protein, human; C489561:Dok1 protein, mouse; D004791:Enzyme Inhibitors; C074561:GAP-associated protein p62; D010750:Phosphoproteins; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D014443:Tyrosine; D005982:Glutathione Transferase; D019869:Phosphatidylinositol 3-Kinases; D019009:Proto-Oncogene Proteins c-kit; C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras); D000077191:Wortmannin