YMXM Motifs and Signaling by an Insulin Receptor Substrate 1 Molecule without Tyrosine Phosphorylation Sites

Abstract

; IRS-1 YCT was tyrosine phosphorylated but also failed to mediate these signaling events. Neither IRS-1 3YMXM nor IRS-1 YCT mediated activation of mitogen-activated protein kinases. IRS-1 F18 and IRS-1 YCT partially mediated similar levels of insulin-stimulated mitogenesis at high insulin concentrations, however, suggesting that IRS-1 contains phosphotyrosine-independent elements which effect mitogenic signals, and that the sites in IRS-1 YCT do not augment this signal. IRS-1 3YMXM mediated the maximal mitogenic response to insulin, although the response to insulin was more sensitive with wild-type IRS-1. By contrast, the association of IRS-1 3YMXM with PI 3-kinase was more sensitive to insulin than the association with IRS-1. Thus, the binding of SH2 proteins (such as PI 3-kinase) by YMXM motifs in IRS-1 is an important element in the mitogenic response, but other elements are essential for full mitogenic sensitivity.The IRS proteins (IRS-1 and IRS-2) are endogenous cellular substrates of the insulin and insulin-like growth factor 1 (IGF-1) receptor tyrosine kinases, as well as substrates for various cytokine receptors which activate various members of the Janus kinase family (22,41,42,47,48). Tyrosine phosphorylation plays a central role in the mechanism of IRS protein signaling by providing docking sites for proteins containing isoforms of the Src homology 2 (SH2) domain (25,38,39). Since the phosphorylation sites in IRS-1 are situated in various amino acid sequence motifs, IRS-1 engages numerous SH2 proteins, including p85, Grb-2, Fyn, Nck, and SHP2 (SHPTP2, Syp, and PTP1D) (10,15,36). IRS proteins contain several phosphorylation sites in YXXM motifs, which are selective binding sites for the SH2 domains in the regulatory subunits (p85 and p55 PIK ) of phosphatidylinositol (PI) 3Ј-kinase (22,28,41). During insulin stimulation, IRS proteins mediate the direct activation of PI 3Ј-kinase, which appears to play essential roles in the activation of the p70 s6k and the akt kinase (21, 22) and the serine phosphorylation of eIF4e and PHAS-I as well as in mediating various biological responses including vesicle movement (including glucose transporter translocation), mitogenesis, general protein synthesis, receptor endocytosis, and chemotaxis, among other things (22,25,48).While the plethora of signaling events apparently mediated by PI 3Ј-kinase raises questions about whether PI 3Ј-kinase is a regulator of specific cellular events or whether it is a global potentiator of cellular signaling, it is obviously an important molecule in the transmission of downstream signals by tyrosine kinases. The 110-kDa PI 3Ј-kinases are coupled to tyrosine kinases by regulatory adapter molecules (p85 and p55 PIK ) which use their SH2 domains to bind tyrosyl-phosphorylated YMXM motifs (14, 28). The 110-kDa catalytic domain (p110) is activated by the binding of these tyrosyl-phosphorylated YMXM motifs (2). In addition to its ability to phosphorylate PI, p110 also phosphorylates certain proteins on serine residues (3,8,14,17). The SH2 d...

c-Jun N-terminal Kinase (JNK) Mediates Feedback Inhibition of the Insulin Signaling Cascade

“…Irs1 , a rat Irs1 mutant that lacks all the known tyrosine phosphorylation sites, including those that activate the PI 3-kinase and ERK pathways (48). Consistent with the inhibitory effect of LY294002 or PD98059 on JNK phosphorylation, insulin failed to stimulate JNK phosphorylation in 32D IR /F18 Irs1 cells (Fig.…”

“…32D cells are murine myeloid progenitors that express few endogenous insulin receptors and no Irs proteins and require interleukin-3 for growth (48); however, they naturally express JNK1 and JNK2 (data not shown). JNK1 and JNK2 are activated by tandem Thr/Tyr phosphorylation (Thr 183 and Tyr 185 in JNK1), which is detected in both isoforms by immunoblotting with a phosphospecific-JNK antibody (␣JNK PI ).…”

The insulin receptor substrate (IRS)-1 recruits phosphatidylinositol 3-kinase to Ret: evidence for a competition between Shc and IRS-1 for the binding to Ret

“…Upon receptor stimulation, IRS-1 is rapidly phosphorylated at many tyrosine positions and engages numerous SH2 proteins including p85, Grb2, Fyn, Nck, phospholipase Cg, and the phosphatase SH-PTP2 . Several phosphorylation sites in YXXM motifs are clustered together in the middle of the IRS-1 protein; accordingly, IRS-1 mediates the direct activation of PI3-K (Myers et al, 1996;Wang et al, 1993). Here we show that IRS-1 binds to the NKLpY(1062) motif of Ret, competes with the binding of Shc to the same docking site, and is involved in Ret-mediated activation of PI3-K.…”

The AP-1 Complex Regulates Intracellular Localization of Insulin Receptor Substrate 1, Which Is Required for Insulin-Like Growth Factor I-Dependent Cell Proliferation

“…7E), indicating that mislocalization of IRS-1 caused by lack of interaction with AP-1 reduced tyrosine phosphorylation of IRS-1 and subsequent Akt phosphorylation. However, it is also possible to ascribe this inhibition to the deletion of tyrosine residues in APBR because Y608, Y628, and Y658 are known to be phosphorylated by IR/IGF-IR and function as binding sites of PI 3-kinase (29,30). To examine whether mislocalization or the deletion of tyrosine residues inhibited the phosphorylation of IRS-1 and subsequent association of PI 3-kinase, we constructed an IRS-1 mutant in which the C terminus is fused with the partial CAAX sequence of H-Ras (IRS-1 CAAX).…”