Stephens L R, Anderson K E, Hawkins P T
Inositide Laboratory, Signalling Programme, The Babraham Institute, Babraham, Cambridge CB2 4AT, United Kingdom.
J Biol Chem. 2001 Nov 16;276(46):42767-73. doi: 10.1074/jbc.M107194200. Epub 2001 Aug 27.
DAPP-1 (dual-adaptor for phosphotyrosine and 3-phosphoinositides-1) is a broadly distributed pleckstrin homology (PH) and Src homology 2 domain containing protein that can bind phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) and can be phosphorylated on tyrosine 139 and internalised in response to activation of type I phosphoinositide 3-kinases (PI3K). Tyrosine phosphorylation of DAPP-1 appears important for appropriate intracellular targeting and creates a potential binding site for Src homology 2 domain-containing proteins. In endothelial cells overexpressing wild-type platelet-derived growth factor beta (PDGFbeta) receptors, which express Bmx and Src as their major Btk (Bruton's tyrosine kinase) family and Src family tyrosine kinases, respectively, PDGF can stimulate PI3K-dependent tyrosine phosphorylation of DAPP-1. Transient overexpression of Src most effectively, compared with Bmx and Syk, augments basal and PDGF-stimulated tyrosine phosphorylation of DAPP-1, whereas overexpression of dominant-negative Src, but not dominant-negative Bmx, inhibits PDGF-stimulated phosphorylation of DAPP-1. Cells expressing mutant PDGFbeta (Y579F/Y581F) receptors (which fail to bind and activate Src-type kinases) fail to tyrosine phosphorylate DAPP-1 in response to PDGF. We show that in DT40 chicken B cell lines, antibody stimulation leads to PI3K-dependent tyrosine phosphorylation of DAPP-1 that is lost in Lyn- or Syk-deficient cell lines but not Btk-deficient cell lines. PI3K-dependent activation of PKB is only lost in Syk-deficient lines. Finally, in vitro we find lipid-modified Src to be the most effective DAPP-1 tyrosine kinase (versus Syk, Lyn, Btk, and Bmx); phosphorylation of DAPP-1 but not Src autophosphorylation is stimulated approximately 10-fold by PtdIns(3,4,5)P(3) (IC(50) = 150 nm) and phosphatidylinositol 3,4-bisphosphate but not by their nonbiological diastereoisomers and depends on PH domain mediated binding of DAPP-1 to PtdIns(3,4,5)P(3)-containing membranes. We conclude that Src family kinases are responsible for tyrosine phosphorylation of DAPP-1 in vivo and that PI3K regulation is at the level of PH domain-mediated translocation of DAPP-1 to PI3K products in the membrane.
DAPP-1（双衔接蛋白，含磷酸酪氨酸和3-磷酸肌醇-1）是一种广泛分布的含有pleckstrin同源结构域（PH）和Src同源结构域2的蛋白质，它能结合磷脂酰肌醇3,4,5-三磷酸（PtdIns(3,4,5)P(3)），并可在酪氨酸139位点发生磷酸化，且在I型磷酸肌醇3激酶（PI3K）激活后被内化。DAPP-1的酪氨酸磷酸化对于其在细胞内的正确定位似乎很重要，并为含Src同源结构域2的蛋白质创造了一个潜在的结合位点。在过表达野生型血小板衍生生长因子β（PDGFβ）受体的内皮细胞中，该受体分别将Bmx和Src作为其主要的Btk（布鲁顿酪氨酸激酶）家族和Src家族酪氨酸激酶表达，PDGF可刺激DAPP-1的PI3K依赖性酪氨酸磷酸化。与Bmx和Syk相比，Src的瞬时过表达最有效地增强了DAPP-1的基础酪氨酸磷酸化和PDGF刺激的酪氨酸磷酸化，而显性负性Src的过表达而非显性负性Bmx的过表达则抑制了PDGF刺激的DAPP-1磷酸化。表达突变型PDGFβ（Y579F/Y581F）受体（无法结合并激活Src型激酶）的细胞在受到PDGF刺激时无法使DAPP-1发生酪氨酸磷酸化。我们发现，在DT40鸡B细胞系中，抗体刺激会导致DAPP-1的PI3K依赖性酪氨酸磷酸化，这种磷酸化在Lyn或Syk缺陷的细胞系中会消失，但在Btk缺陷的细胞系中不会。PI3K依赖性的蛋白激酶B（PKB）激活仅在Syk缺陷的细胞系中消失。最后，在体外我们发现脂质修饰的Src是最有效的DAPP-1酪氨酸激酶（与Syk、Lyn、Btk和Bmx相比）；PtdIns(3,4,5)P(3)（IC(50)=150 nM）和磷脂酰肌醇3,4-二磷酸可刺激DAPP-1的磷酸化，但不刺激Src的自身磷酸化，且刺激作用约为10倍，而非生物非对映异构体则无此作用，并且这取决于PH结构域介导的DAPP-1与含PtdIns(3,4,5)P(3)的膜的结合。我们得出结论，Src家族激酶在体内负责DAPP-1的酪氨酸磷酸化，并且PI3K调节作用发生在PH结构域介导的DAPP-1向膜中PI3K产物的转位水平。
