Phosphoinositide 3-kinase-dependent phosphorylation of the dual adaptor for phosphotyrosine and 3-phosphoinositides by the Src family of tyrosine kinase.
We recently identified a novel adaptor protein, termed dual adaptor for phosphotyrosine and 3-phosphoinositides (DAPP1), that possesses a Src homology (SH2) domain and a pleckstrin homology (PH) domain. DAPP1 exhibits a high-affinity interaction with PtdIns(3,4,5)P(3) and PtdIns(3,4)P(2), which bind to the PH domain. In the present study we show that when DAPP1 is expressed in HEK-293 cells, the agonists insulin, insulin-like growth factor-1 and epidermal growth factor induce the phosphorylation of DAPP1 at Tyr(139). Treatment of cells with phosphoinositide 3-kinase (PI 3-kinase) inhibitors or expression of a dominant-negative PI 3-kinase prevent phosphorylation of DAPP1 at Tyr(139), and a PH-domain mutant of DAPP1, which does not interact with PtdIns(3,4,5)P(3) or PtdIns(3,4)P(2), is not phosphorylated at Tyr(139) following agonist stimulation of cells. Overexpression of a constitutively active form of PI 3-kinase induced the phosphorylation of DAPP1 in unstimulated cells. We demonstrated that Tyr(139) of DAPP1 is likely to be phosphorylated in vivo by a Src-family tyrosine kinase, since the specific Src-family inhibitor, PP2, but not an inactive variant of this drug, PP3, prevented the agonist-induced tyrosine phosphorylation of DAPP1. Src, Lyn and Lck tyrosine kinases phosphorylate DAPP1 at Tyr(139) in vitro at similar rates in the presence or absence of PtdIns(3,4,5)P(3), and overexpression of these kinases in HEK-293 cells induces the phosphorylation of Tyr(139). These findings indicate that, following activation of PI 3-kinases, PtdIns(3,4,5)P(3) or PtdIns(3,4)P(2) bind to DAPP1, recruiting it to the plasma membrane where it becomes phosphorylated at Tyr(139) by a Src-family tyrosine kinase.
Dowler
S
S
MRC Protein Phosphorylation Unit, Department of Biochemistry, MSI/WTB Complex, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, UK. s.j.dowler@dundee.ac.uk
Montalvo
L
L
Cantrell
D
D
Morrice
N
N
Alessi
D R
DR
D048868:Adaptor Proteins, Signal Transducing; D001798:Blood Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli; D005227:Fatty Acids / Q000378:metabolism*; D006801:Humans; D008074:Lipoproteins; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000096:biosynthesis / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010716:Phosphatidylinositols / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D019000:Phosphotyrosine / Q000378:metabolism; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D001798:Blood Proteins; D004791:Enzyme Inhibitors; D005227:Fatty Acids; D008074:Lipoproteins; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D019869:Phosphatidylinositol 3-Kinases; D010716:Phosphatidylinositols; D019000:Phosphotyrosine; D019061:src-Family Kinases; C486555:DAPP1 protein, human; C060902:lyn protein-tyrosine kinase