Search

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 (Tyr114) in PHB is phosphorylated in response to insulin stimulation. In addition, using active insulin receptor (IR) and synthetic biotinylated PHB peptide (PHB107–121) we have shown that IR also phosphorylates Tyr114 in an in vitro kinase assay. Phosphorylation of PHB at Tyr114 was confirmed by immunoblotting using anti-phosphoTyr114 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-PHB107–121 peptide phosphorylated at Tyr114 was also able to pull down Shp1 in pull down assays. Non-phosphorylated PHB107–121 peptide, corresponding PHB2121–135 peptide and Tyr114Phe mutant-PHB fail to pull down Shp1. In summary, we have identified Tyr114 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. [Copyright &y& Elsevier]

The Src homology 2 (SH2) domain of interleukin-2 tyrosine kinase (Itk) is a critical component of the regulatory apparatus controlling the activity of this immunologically important enzyme. To gain insight into the structural features associated with the activated form of Itk, we have solved the NMR structure of the SH2 domain bound to a phosphotyrosine-containing peptide (pY) and analyzed changes in trans-hydrogen bond scalar couplings (3h J NC′) that result from pY binding. Isomerization of a single prolyl imide bond in this domain is responsible for simultaneous existence of two distinct SH2 conformers. Prolyl isomerization directs ligand recognition: the trans conformer preferentially binds pY. The structure of the SH2/pY complex provides insight into the ligand specificity; the BG loop in the ligand-free trans SH2 conformer is pre-arranged for optimal contacts with the pY+3 residue of the ligand. Analysis of 3h J NC′ couplings arising from hydrogen bonds has revealed propagation of structural changes from the pY binding pocket to the CD loop containing conformationally heterogeneous proline as well as to the αB helix, on the opposite site of the domain. These findings offer a structural framework for understanding the roles of prolyl isomerization and pY binding in Itk regulation. [Copyright &y& Elsevier]