Insulin Receptor Substrate Proteins Create a Link between the Tyrosine Phosphorylation Cascade and the Ca2+-ATPases in Muscle and Heart

Abstract

Following phosphorylation by the insulin receptor kinase, the insulin receptor substrates (IRS)-1 and IRS-2 bind to and activate several Src homology 2 (SH2) domain proteins. To identify novel proteins that interact with IRS proteins in muscle, a human skeletal muscle cDNA expression library was created in the EXlox system and probed with baculovirus-produced and tyrosine-phosphorylated human IRS-1. One clone of the 10 clones which was positive through three rounds of screening represented the C terminus of the human homologue of the adult fast twitch skeletal muscle Ca 2؉ -ATPase (SERCA1) including the cytoplasmic tail and part of transmembrane region 10. Western blot analysis of extracts of rat muscle demonstrated co-immunoprecipitation of both IRS-1 and IRS-2 with the skeletal muscle Ca 2؉ -ATPase (SERCA1) and the cardiac muscle isoform (SERCA2). In both cases, injection of insulin stimulated a 2-to 6-fold increase in association of which was maximal within 5 min. In primary cultures of aortic smooth muscle cells and C2C12 cells, the insulin-stimulated interaction between IRS proteins and SERCA1 and -2 was dose-dependent with a maximum induction at 100 nM insulin. This interaction was confirmed in a "pull down" experiment using a glutathione S-transferase fusion protein containing the C terminus of the human SERCA isoform and phosphorylated IRS-1 in vitro and could be blocked by a FLVRES-like domain peptide present in the human SERCA sequence. Affinity chromatography of phosphopeptide libraries using the glutathione S-transferase fusion protein of the C terminus of SERCA1 indicated a consensus sequence for binding of XpYGSS; this is identical to potential tyrosine phosphorylation sites at position 431 of human IRS-1 and at position 500 of human IRS-2. In streptozotocin diabetic rats the interaction between IRS proteins and SERCA1 in skeletal muscle and SERCA2 in cardiac muscle was significantly reduced. Taken together, these results indicate that the IRS proteins bind to the Ca 2؉ -ATPase of the sarcoplasmic reticulum in an insulin-regulated fashion, thus creating a potential link between the tyrosine phosphorylation cascade and effects of insulin on calcium.Insulin binding to the insulin receptor leads to the tyrosine phosphorylation of several insulin receptor substrates (IRS), 1 including IRS-1, IRS-2, IRS-3, Gab1, and Shc (1-3). These insulin receptor substrates then bind to a number of proteins in the cell via interaction of tyrosine phosphorylation motifs and specific domains of the target proteins termed SH2 (src homology 2) domains (4 -6). For IRS-1 and IRS-2, the SH2 proteins include the lipid-modifying enzyme phosphatidylinositol (PI) 3-kinase, the cytoplasmic tyrosine kinase Fyn, the protein tyrosine phosphatase SHP2, and the adaptor molecules Nck and Grb2 (7-12). Exactly how IRS binding to SH2 domain proteins results in signal transduction is still not understood, but for PI 3-kinase and SHP2, binding of phosphorylated IRS-1 results in stimulation of enzymatic activity (13,14). Grb2, on the other ...

Increased red cell sodium-lithium countertransport and lymphocyte cytosolic calcium are separate phenotypes in patients with essential hypertension

“…Insulin stimulates both the plasma membrane Ca-ATPase and the sarcoplasmic (endoplasmic) reticulum Ca-ATPase. [21][22][23] Insulininduced increases in the activity of either pathway could be associated with lower intracellular calcium in insulin sensitive patients. In support of this hypothesis, we have observed that hypertensives with high Ca cyt have a substantial fall in Ca cyt with insulin incubation in vitro (unpublished observations).…”

Insulin Receptor Substrate 1-induced Inhibition of Endoplasmic Reticulum Ca2+ Uptake in β-Cells

“…This is also strongly supported by the observed decrease in ␤IRS1-A ER Ca 2ϩ uptake (an indirect measurement of Ca 2ϩ -ATPase) and the fact that Ca 2ϩ release from the ER (induced by A23187, data not shown) is not changed in ␤IRS1-A cells. A possible explanation for IRS-1-induced inhibition of ER Ca 2ϩ -ATPase is based on an elegant study by Kahn and co-workers (33), who have identified SERCA1 in skeletal muscle and SERCA2 in cardiac muscle as novel IRS-1-and IRS-2-binding proteins. Importantly, this interaction is insulin-dependent (maximal at 100 nM insulin and at 5 min of stimulation), and requires tyrosine phosphorylation of IRS-1.…”

“…Addition of thapsigargin to pancreatic ␤-cells leads to elevated cytosol Ca 2ϩ concentration and enhanced short term glucose-stimulated insulin secretion (32). Recent data showed that IRSs may directly interact with ER Ca 2ϩ -ATPase (SERCA1 and SERCA2) in a tyrosine phosphorylation-dependent manner in muscle and heart (33). This finding suggests that insulin may via insulin receptor signaling pathway regulate ER Ca 2ϩ -ATPase activity, therefore influencing cellular homeostasis and ␤-cell dysfunction.…”

Dissection of the insulin signaling pathway via quantitative phosphoproteomics

“…With our pull down strategy, we identified the myosin regulatory light chain 2 as an interactor of the PISP/PDZK11 protein, and this may be a link to Ca 2ϩ dependent phosphorylation and glucose uptake. Previously, the sarcoplasmatic Ca 2ϩ -ATPases SERCA1 and SERCA2, have been shown to interact with the IRS proteins (40). Using a recently developed peptide pull-down approach based on SILAC, we identified SERCA2 and the S-100 calcium binding protein as specific binding partners of the tyrosine phosphorylated PISP/PDZK11.…”