Phosphorylation Effects on Flanking Charged Residues

Abstract

*H-NMR and 31P-NMR spectroscopy were employed to assess the electrostatic consequences of phosphorylation of single and multiple tyrosine residues in peptides derived from the core and tail autophosphorylation regions of the human insulin receptor tyrosine-kinase domain. In both peptides, phosphorylation was accompanied by changes in the resonances from basic side-chains ; those from acidic residues were unaffected. Tyrosine phosphorylation caused increases of up to one in the pK, values of histidine residues situated up to eight residues away in the primary sequence. Titration curve analysis by Hill plots suggested some cooperativity of histidine and phosphate ionizations. Behaviour closely analogous to that of the insulin receptor tail peptide was observed during changes in phosphorylation of the intact insulin receptor kinase domain, suggesting that the electrostatic dissemination effects seen for the isolated peptide are retained by the peptide sequence in the context of the much larger protein. Similar changes in the behaviour of basic residues were also observed upon tyrosine phosphorylation of a cdc2-derived peptide, suggesting that this potential of phosphorylation events to propagate directed structural changes may find a widespread utility in the activation of protein kinases and in the transduction of phosphorylation-based signalling.Reversible protein phosphorylation is the most common cellular regulation mechanism. Phosphorylation is associated with a stereochemical influence on the equilibrium distribution of conformational states of the protein, which results in a net change in the average protein structure. Two mechanisms of phosphoregulation have so far been recognised: allosteric regulation in phosphorylase, where phosphorylation induces a domain rotation (reviewed by Johnson, 1992), and, as exemplified by isocitrate dehydrogenase, direct control mediated principally by electrostatic effects at the active site, thereby modulating substrate binding (Hurley et al., 1989).The recently determined structure of a phosphoregulated kinase (ERK2) (Zhang et al., 1994) suggests that a combination of these two paradigm mechanisms underlies the regulation of catalytic activity common to many kinases, a large number of which require multisite phosphorylation to trigger kinase activity. One such example is the human insulin receptor Correspondence to B. A. Levine,

Autophosphorylation of Type Iβ cGMP-dependent Protein Kinase Increases Basal Catalytic Activity and Enhances Allosteric Activation by cGMP or cAMP

“…Thus, autophosphorylation must also overcome these additional sites of interaction. Using synthetic peptides, it was recently demonstrated that phosphorylation alters the charge of basic residues that flank the phosphoacceptor (53). This effect, in combination with repulsion of the catalytic domain or other components of the regulatory domain, and a reduction in the hydrophobicity of the pseudosubstrate region could provide a potent force to induce conformational changes resulting in activation of the enzyme.…”

Conformational Changes in Protein Loops and Helices Induced by Post-Translational Phosphorylation

“…Structural information is particularly limited, due in part to the difficulty of obtaining sufficient purified protein in a specific modification state. X-ray crystallography has determined atomic-resolution structures for a few tens of phosphorylated proteins [3], whereas nuclear magnetic resonance (NMR) experiments have elucidated structure and dynamics of a similar number of phosphorylated peptides and small proteins in solution [4][5][6][7][8][9][10]. Electron paramagnetic resonance (EPR) and circular dichroism experiments can also provide information on conformational change due to phosphorylation [11], but do not provide atomic detail.…”

Ras binding to a C‐terminal region of GAP

“…1) of the sequence of human p120 CaP were prepared, deblocked and purified to homogeneity by HPLC using a Vydac Cjs column eluted with a linear CH3CN gradient and lyophilised as described [21]. In order to rule out disulphide bridge formation and at the same time introduce environmentally sensitive extrinsic fluorescent chromophores, Y922 was carbox-ymethylated at the -SH group of C745 with either IAEDANS (Y922.IAEDANS) or IAS (Y922.IAS).…”