Insulin induced phosphorylation of prohibitin at tyrosine114 recruits Shp1

Abstract

Prohibitin (PHB or PHB1) is an evolutionarily conserved ubiquitously expressed multifunctional protein and is present in various cellular compartments. Phosphorylation of PHB has been suggested as one of the potential mechanisms in the regulation of its various functions however exact sites of phosphorylation remain to be determined. To better understand the functional relevance of phosphorylation of PHB, we have explored the potential sites of phosphorylation using combination of approaches including phosphoamino specific immunoblotting, proteolysis, two-dimensional gel electrophoresis, phosphoamino acid analysis and site-directed mutagenesis techniques and report that tyrosine 114 (Tyr 114) in PHB is phosphorylated in response to insulin stimulation. In addition, using active insulin receptor (IR) and synthetic biotinylated PHB peptide (PHB(107-121)) we have shown that IR also phosphorylates Tyr 114 in an in vitro kinase assay. Phosphorylation of PHB at Tyr 114 was confirmed by immunoblotting using anti-phosphoTyr 114 specific antibody. Furthermore, we demonstrate that SH2 domain containing tyrosine phosphatase-1 (Shp1) co-immunoprecipitate with PHB antiserum after insulin induced phosphorylation of PHB. Biotinylated-PHB(107-121) peptide phosphorylated at Tyr 114 was also able to pull down Shp1 in pull down assays. Non-phosphorylated PHB(107-121) peptide, corresponding PHB2(121-135) peptide and Tyr114Phe mutant-PHB fail to pull down Shp1. In summary, we have identified Tyr 114 in PHB as an important site of phosphorylation and phosphorylation at this residue creates a binding site for Shp1 both in vivo and in vitro.

Prohibitin-2 Binding Modulates Insulin-like Growth Factor-binding Protein-6 (IGFBP-6)-induced Rhabdomyosarcoma Cell Migration

“…The PHB1⅐PHB2 phosphocomplex is required for mitochondrial homeostasis and survival of human T cells (47). Insulin-induced phosphorylation of cell membrane PHB1 at tyrosine 114 and 259 by insulin receptor kinase facilitates its interaction with Shp1, which in turn negatively regulates PIP3/Akt signaling in myoblasts (48) and breast cancer cells (48,49). On the other hand, Akt-induced threonine 258 phosphorylation of PHB1 inhibits its interactions with PIP3 and Shp2, which may facilitate insulin-induced PIP3/Akt signaling in 3T3-L1 adipocytes (50).…”

Recent insights into the actions of IGFBP-6

“…Phosphorylation of prohibitins modulated their effects on cell signalling and function (Mishra et al 2010). Insulin phosphorylated PHB1 at Tyr114, which is conserved in PHB2, resulting in SHP1 recruitment and decreased AKT phosphorylation (Ande, et al 2009). …”

Prohibitin has an important role in adipocyte differentiation

“…23,31 The following primers were used to generate IRE mutant (mIRE) reporter constructs: À1031/À1025 mutant, forward 5 0 -C GTCTCTACAAAAATAAAAAATTAGCCGGGTGTCCCCCCGCATGCCTGTAATCTCA GCTATTCTG-3 0 reverse 5 0 -CAGAATAGCTGAGATTACAGGCATGCGGGGGGA CACCCGGCTAATTTTTTATTTTTGTAGAGACG-3 0 and À1010/À1004 mutant,…”

“…24,27,36 Furthermore, we have shown that phosphorylation of PHB at tyrosine114 (Tyr114) residue attenuates PI3K/Akt signaling. 23,24 In this study, we tested whether forced expression of PHB would change insulin-induced adipogenesis. To determine this, we used 3T3-L1 fibroblasts as a model cell line.…”

“…Recently, we have reported that PHB modulates insulin signaling in a phosphorylation-dependent manner. 23,24 In addition, we have shown that PHB interacts with pyruvate carboxylase (PC), 25 an important enzyme involved in de novo fatty acid synthesis and glyceroneogenesis. 26 Furthermore, PHB has been reported to be involved in Raf activation and to have an important role in MAPK/ERK signaling.…”