DAPP1 undergoes a PI 3-kinase-dependent cycle of plasmamembrane recruitment and endocytosis upon cell stimulation

Background: Phosphoinositide (PI) 3-kinase and its second messenger product s, phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P-3) and phosphat iclylinositol 3,4-bisphosphate (PtdIns(3,4)P-2), play important roles in si gnalling processes crucial for cell movement, differentiation and survival. Previously, we isolated a 32 kDa PtdIns(3,4,5)P-3-binding protein from por cine leukocytes. This protein contains an amino-terminal Src homology 2 (SH 2) domain and a carboxy-terminal pleckstrin homology (PH) domain, and is id entical to the recently described DAPP1 (also known as PHISH or Bam32) prot ein. Here, we characterised the subcellular distribution of DAPP1 in respon se to cell stimulation, Results: When expressed transiently in porcine aortic endothelial (PAE) cel ls, DAPP1 translocated from the cytosol to the plasma membrane in response to platelet-derived growth factor (PDGF). This translocation was dependent on both P1 3-kinase activity and an intact DAPP1 PH domain. Following recru itment to the plasma membrane, DAPP1 entered the cell in vesicles, Similar responses were seen in DT40 chicken B cells following antibody treatment, a nd Rat-1 fibroblasts following epidermal growth factor (EGF) or PDGF treatm ent. Colocalisation studies in PAE cells suggested entry of DAPP1 by endocy tosis in a population of early endosomes containing internalised PDGF-beta receptors. DAPP1 also underwent PI 3-kinase-dependent phosphorylation on Ty r1 39 in response to PDGF stimulation, and this event was involved in the v esicular response. Conclusions: This is the first report of plasma-membrane recruitment and en docytosis of a PI 3-kinase effector protein in response to cell stimulation . The results suggest a novel role for DAPP1 in endosomal trafficking or so rting.