Adapter protein with a pleckstrin homology (PH) and an Src homology 2 (SH2) domain (APS) and SH2-B are adapter proteins and substrates that interact with the activation loop of the insulin-receptor (IR) kinase. These proteins are homologous and share substantial sequence similarity. We previously showed [Ahmed, Smith and Pillay, FEBS Lett. 475, 31–34], for the first time, that insulin-stimulated phosphorylation of APS led to interaction with c-Cbl in 3T3-L1 adipocytes and in transfected Chinese-hamster ovary (CHO) cells. In the present study, we find that insulin stimulates the membrane translocation and phosphorylation of APS to a much greater extent than SH2-B, despite the structural similarity of these proteins. Expression of APS or SH2-B delays IR tyrosine and IR substrate (IRS) dephosphorylation. This enhancement of signalling is also observed downsteam of the receptor. In control cells that lack APS, following insulin stimulation, extracellular-signal-regulated kinase (ERK) and Akt kinase reach maximal activation and then decline to basal levels by 60min. In contrast, in APS- and SH2-B-expressing cells, ERK and Akt kinase activation remains at peak levels at 60min. These effects may occur because these proteins either stabilize the active conformation or prevent dephosphorylation of the IR. We therefore conclude that, despite the ability to couple to c-Cbl, APS functions as a positive regulator of IR signalling and, although SH2-B is a poor substrate for the IR, its association with the IR allows it to regulate pathways downstream of the receptor independently of its phosphorylation.
Abbreviations used: APS, adapter protein with a PH and an SH2 domain; CHO, Chinese-hamster ovary; CHO.T, IR-transfected CHO; ERK, extracellular-signal-regulated kinase; FCS, foetal calf serum; GLUT4, glucose transporter 4; Grb2, growth-factor-receptor-bound protein 2; IR, insulin receptor; IRS, IR substrate; JAK, Janus kinase; MEK, mitogen-activated protein kinase/ERK kinase; PH, pleckstrin homology; PI3K, phosphoinositide 3-kinase; PTP, phosphotyrosine phosphatase; SH2, Src homology 2; SHC, SH2-domain-containing and collagen; SOS, son of sevenless.
Present address: Department of Biochemistry and Molecular Biology, Darwin Building, University College London, Gower Street, London WC1E 6BT, U.K.