Regulation of Platelet-derived Growth Factor Receptor Activation by Afadin through SHP-2

Abstract

Upon binding of platelet-derived growth factor (PDGF), PDGF receptor is autophosphorylated at tyrosine residues in its cytoplasmic region, which induces the activation of diverse intracellular signaling pathways such those involving Ras-ERK, c-Src, and Rap1-Rac. Signaling through activated Ras-ERK promotes cell cycle and cell proliferation. The sequential activation of Rap1 and Rac affects cellular morphology and induces the formation of leading-edge structures, including lamellipodia, peripheral ruffles, and focal complexes, resulting in the enhancement of cell movement. In addition to the promotion of cell proliferation, the Ras-ERK signaling is involved in the regulation of cellular morphology. Here, we showed a novel role of afadin in the regulation of PDGF-induced intracellular signaling and cellular morphology in NIH3T3 cells. Afadin was originally identified as an actin filament-binding protein, which binds to a cell-cell adhesion molecule nectin and is involved in the formation of cell-cell junctions. When afadin was tyrosine-phosphorylated by c-Src activated in response to PDGF, afadin physically interacted with and increased the phosphatase activity of Src homology 2 domain-containing phosphatase-2 (SHP-2), a protein-tyrosine phosphatase that dephosphorylates PDGF receptor, leading to the prevention of hyperactivation of PDGF receptor and the Ras-ERK signaling. In contrast, knockdown of afadin or SHP-2 induced the hyperactivation of PDGF receptor and Ras-ERK signaling and consequently suppressed the formation of leading-edge structures. Thus, afadin plays a critical role in the proper regulation of the PDGF-induced activation of PDGF receptor and signaling by Ras-ERK. This effect, which is mediated by SHP-2, impacts cellular morphology.Afadin is a nectin-and actin filament (F-actin) 2 -binding protein that connects nectin to the actin cytoskeleton (1). Nectin is a Ca 2ϩ -independent immunoglobulin-like molecule that first forms cell-cell adhesion and then assembles cadherin at the nectin-based cell-cell adhesion sites, resulting in the formation of adherens junctions (AJs) in epithelial cells and fibroblasts (2, 3). The nectin family of proteins comprises four members, nectin-1, -2, -3, and -4. A series of our studies revealed the roles and modes of action of nectin and afadin for the formation of AJs (2-10). Nectin initiates the formation of cell-cell adhesion and then induces the activation of Rap1, Cdc42, and Rac small G proteins through the activation of c-Src. Subsequently, Cdc42 and Rac bind to IQGAP1 and induce the reorganization of the actin cytoskeleton, thereby causing the accumulation of nontrans-interacting E-cadherin at the nectin-based cell-cell adhesion sites. On the other hand, afadin, which binds to Rap1 activated by the action of nectin, associates with p120 ctn and inhibits the endocytosis of accumulated non-trans-interacting E-cadherin to facilitate the trans-interaction of E-cadherin. In parallel with these processes, Cdc42 increases the number of filopodia and cell-cell contact si...

Role of Afadin in Vascular Endothelial Growth Factor– and Sphingosine 1-Phosphate–Induced Angiogenesis

“…We demonstrated here that afadin plays a critical role in proangiogenic activities of ECs and in vivo angiogenesis and clarified how afadin regulates endothelial signaling downstream of Rap1. Because we have reported that afadin regulates migration and survival in fibroblasts 13,18,26,31 and cell-cell adhesion in epithelial cells, 14,32 the critical roles of afadin in migration, survival and cell-cell adhesion in ECs are consistent with our previous reports. However, the role of afadin in tubulogenesis has not been investigated.…”

“…16, 17 We have demonstrated that afadin regulates cell adhesion and polarity in epithelial cells and neurons, and cell movement of fibroblasts. 14,18,19 The roles of afadin in tubulogenesis and angiogenesis are unknown, but the current literature implies afadin may be a promising candidate protein linking Rap1 to angiogenesis. In this study, we therefore attempt to clarify the roles of Rap1 and afadin in the VEGF-and sphingosine 1-phosphate (S1P)-induced angiogenesis.…”

SH2 Domain–Containing Phosphatase-2 Is a Novel Antifibrotic Regulator in Pulmonary Fibrosis

“…Activating mutations in SHP2 underlie the Noonan syndrome that manifests as congenital heart disease, short stature, and facial dysmorphia (15,16). SHP2 regulates activated tyrosine kinase receptors including PDGFR, by binding to and dephosphorylating them (17,18). In mice targeted deletions of SHP2 in alveolar epithelial cells (AECs) (19) or macrophages (20) increase predisposition to fibrosis, but the expression of SHP2 in human pulmonary fibrosis or its potential therapeutic usefulness are still unknown.…”

Endothelial and Smooth Muscle-derived Neuropilin-like Protein Regulates Platelet-derived Growth Factor Signaling in Human Vascular Smooth Muscle Cells by Modulating Receptor Ubiquitination

“…Existing data suggest that these effects are mediated by PDGFR␤, but not PDGFR␣, signaling as the latter cannot be activated by PDGF-BB (7). PDGF binding to PDGFR␤ leads to receptor dimerization, autophosphorylation, and recruitment of Src homology 2 (SH2) domain-containing signaling molecules (16). This in turn leads to MAPK (ERK1/2, p38 MAPK, and stress-activated protein kinase/c-Jun N-terminal kinase) activation (17).…”