Browsing by Subject "IRS2"

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Identification and functional studies of two novel serine phosphorylation sites of insulin receptor substrate (IRS)-2: Ser 675 and Ser 907
(2010) Fritsche, Louise; Schleicher, Erwin D.
Insulin receptor substrate (IRS) proteins are major transducers of the insulin and IGF-1 signal into the PI-3 kinase/PKB and the MAP kinase pathway. In addition to tyrosine phosphoryla-tion, a large number of serine/threonine phosphorylation sites enable the IRS proteins to inte-grate different extra- and intracellular stimuli resulting in positive and negative modulation of the insulin and IGF-1 signal. Chronic hyperphosphorylation of serine/threonine sites of IRS-1 is involved in the development of insulin resistance. IRS-2 is of great importance for β-cell survival and for the regulation of hepatic metabolism. The study of serine/threonine phos-phorylations is required to understand the physiological and pathophysiological regulation of this important mediator of insulin signaling. In this thesis two novel IRS-2 serine phosphoryla-tion sites have been identified and characterized (mouse amino acid numbering): Ser 675, which is located in the kinase regulatory loop binding (KRLB) domain unique to IRS-2 and Ser 907, which is adjacent to the Grb2 binding site Tyr 911. Using phospho-site specific antibod-ies both sites were demonstrated to be phosphorylated upon insulin, phorbol ester and ani-somycin treatment in Fao rat hepatoma cells. The phosphorylation was also detected in pri-mary human hepatocytes and in liver tissue of insulin treated or refed mice. The insulin-induced phosphorylation of Ser 907 was mediated by the MAP kinase ERK1/2. Simulation of a permanent phosphorylation of this site in BHK cells expressing IRS-2 Glu 907 led to a slight decrease of IRS-2 tyrosine phosphorylation with no apparent effect on insulin downstream signaling. The insulin-induced association of IRS-2 with Grb2 in HEK293 cells was abrogated by mutation of the adjacent Tyr 911 to Phe, but not influenced by mutation of Ser 907 to Ala. Of note, the activation of MAP kinase signaling was not impaired in HEK293 cells expressing IRS-2 Phe 911 and not regulated by the expression level of IRS-2 wildtype, but completely dependent on IR expression, indicating the importance of an alternative, IRS-2-Grb2-independent pathway for the activation of MAP kinase signaling in these cells. The insulin-induced phosphorylation of Ser 675 was dependent on mTOR, but not on the downstream kinase p70 S6K1. Prevention of this phosphorylation in BHK cells or HEK293 cells expressing IRS-2 Ala 675 had no effect on proximal or distal insulin signal transduction. But compared with IRS-2 wildtype, the mutated IRS-2 protein Ala 675 showed increased half life in cycloheximide-treated HEK293 cells. Thus, phosphorylation of Ser 675 could have a similar function as its homologous site Ser 632 in IRS-1 and could be involved in the regula-tion of mTOR-dependent IRS-2 proteasom-mediated protein degradation.
Novel serine phosphorylation sites of IRS2 mediate 14-3-3 binding and regulate insulin signal transduction
(2012) Neukamm, Sabine Sarah; Schleicher, Erwin D.
Insulin and insulin like growth factor (IGF)-1 mediate their metabolic and mitogenic effects on target tissues through activation of the insulin and IGF-1 receptor. Insulin receptor substrate (IRS) proteins function as intermediate docking platforms to transduce the insulin/IGF-1 signal to intracellular effector molecules that regulate glucose homoeostasis, lipid metabolism, cell proliferation and ß-cell survival. The activated receptors function as tyrosine kinases that phosphorylate IRS proteins on tyrosine residues, thereby generating interaction motifs for Src homology (SH) 2 domain containing proteins. Signal transduction via IRS proteins is furthermore regulated by their serine/threonine phosphorylation by several kinases and this can result in inhibitory or stimulatory consequences for downstream signalling. Hyperphosphorylation of IRS1 has been shown to be involved in the desensitization process that can result in insulin resistance. Both IRS1 and IRS2 undergo proteasomal degradation while particularly IRS2 levels are additionally regulated by cAMP-dependent gene activation. 14-3-3 proteins are versatile regulators of a variety of intracellular processes like control of cell cycle, cell growth, gene transcription and apoptosis. Serine/threonine phosphorylation within distinct motifs on the interaction partner is a prerequisite for 14-3-3 binding. IRS1 and IRS2 have been described as 14-3-3 interaction proteins and interaction of IRS2 with 14-3-3 proteins was specifically characterized in this thesis. Insulin/IGF-1-dependent PI 3-kinase stimulation as well as elevated cAMP levels were identified to modulate 14-3-3 binding to IRS2. IGF-1 stimulation led to increased binding of 14-3-3 to IRS2 in transfected HEK293 cells and this bind- ing was prevented by inhibition of the PI 3-kinase pathway and an Akt/PKB inhibitor. Insulin-stimulated interaction between endogenous IRS2 and 14-3-3 was observed in rat hepatoma cells and in mice liver after an acute insulin stimulus or refeeding. Application of different IRS2 fragments enabled localization of the IGF-1-dependent 14-3-3 binding region spanning amino acids 300-600. Mass spectrometric analysis produced a total of 24 phosphorylated serine/threonine residues on IRS2 after IGF-1 stimulation with 12 sites unique for IRS2 while the other residues are conserved in IRS1 and IRS2. The 24 identified phosphorylated residues on IRS2 included several 14-3-3 binding candidates in the region 300-600 and single alanine mutants of these candidates led to the identification of Ser573 as 14-3-3 binding site by overlay assays. A phosphosite specific antibody was generated to further characterize Ser573. IGF-1-dependent phosphorylation of Ser573 was reduced by inhibition of PI 3-kinase and Akt/PKB. The alanine mutant of Ser573 showed enhanced phosphorylation of Akt/PKB in an IGF-1 time course experiment. In summary, the data presented in this thesis indicate a negative impact of Ser573 phosphorylation on downstream signalling. Binding of 14-3-3 to IRS2 upon stimulation with forskolin and the cAMP analogue CPT-cAMP (8-(4-chlorophenylthio) adenosine 3?,5?-cyclic monophosphate) was demonstrated in HEK293 cells, that was prevented with the PKA inhibitor H89. The amino acid region behind position 952 on IRS2 was identified as cAMP/PKA-dependent 14-3-3 binding region by GST-14-3-3 pulldown as- says. Mass spectrometric analyses revealed Ser1137/Ser1138 as cAMP-dependent, potential PKA phosphorylation sites. Inhibition of Akt/PKB or ERK did not prevent the cAMP-dependent phosphorylation of IRS2 on PKA consensus motifs. Mutation of Ser1137/Ser1138 to alanine strongly reduced the cAMP-dependent 14-3-3 binding as shown by GST-14-3-3 pulldown experiments and co-immunoprecipitation assays. IRS2 protein degradation was demonstrated by the application of cycloheximide and an increased IRS2 protein stability was observed when HEK293 cells stably expressing IRS2 or primary hepatocytes were incubated with forskolin. This reduced IRS2 protein degradation was dependent on the presence of Ser1137/Ser1138, since stimulation with forskolin did not increase protein stability of the double Ala1137/Ala1138 mutant. To conclude, Ser1137/Ser1138 are presented as novel cAMP-dependent phosphorylation sites on IRS2 and their importance in 14-3-3 binding and IRS2 protein stability is demonstrated. This represents a novel mechanism for the cAMP- dependent upregulation of IRS2 protein levels that can be of importance for hepatic metabolism and ß-cell survival.