Analysis of the in vivo phosphorylation state of protein phosphatase inhibitor-2 from rabbit skeletal muscle by fast-atom bombardment mass spectrometry.
A new procedure has been developed for identifying phosphoserine residues in proteins, and is used to analyse the in vivo phosphorylation state of inhibitor-2. The method employs reverse-phase liquid chromatography to resolve phosphorylated and dephosphorylated forms of peptides and fast-atom bombardment mass spectrometry (FABMS) to identify phosphorylated derivatives. The positions of phosphorylation sites within peptides are located by gas-phase sequencer analysis after conversion of phosphoserine residues to S-ethylcysteine. The phosphorylation sites on inhibitor-2 were identified as serines-86, -120 and -121, the three residues phosphorylated in vitro by casein kinase-II. Serine-86 was phosphorylated to 0.7 mol/mol and serines-120 and -121 each to 0.3 mol/mol. These values were not altered significantly by intravenous injection of adrenalin or insulin. No phosphate was present in the region comprising residues 1-49, even after injection of adrenalin, demonstrating that inhibitor-2 is not a substrate for cyclic AMP-dependent protein kinase in vivo. The absence of phosphotyrosine also indicated that inhibitor-2 is not a physiological substrate for the insulin receptor. Surprisingly, no phosphate was present at threonine-72, the residue phosphorylated in vitro by glycogen synthase kinase-3, after injection of either propranolol, adrenalin or insulin. The implications of this finding for the in vivo activation of protein phosphatase 1I (the 1:1 complex between inhibitor-2 and the catalytic subunit of protein phosphatase-1) are discussed. FABMS analysis of inhibitor-2 confirmed the accuracy of the primary structure reported previously, and showed that the only post-translational modifications were an N-acetyl moiety and the three phosphoserine residues. FABMS also demonstrated the presence of an additional serine residue at the C-terminus, and showed that 50% of isolated inhibitor-2 molecules lack the C-terminal Ser-Ser dipeptide.
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004791:Enzyme Inhibitors / Q000378:metabolism*; D004837:Epinephrine / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology; D013058:Mass Spectrometry; D009124:Muscle Proteins / Q000378:metabolism; D009842:Oligopeptides / Q000032:analysis; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D010769:Phosphothreonine / Q000378:metabolism; D011433:Propranolol / Q000494:pharmacology; D054645:Protein Phosphatase 1; D011506:Proteins / Q000378:metabolism*; D011817:Rabbits; D000097847:Protein Phosphatase Inhibitory Proteins
D004791:Enzyme Inhibitors; D004837:Epinephrine; D007328:Insulin; D009124:Muscle Proteins; D009842:Oligopeptides; D010749:Phosphoprotein Phosphatases; D010750:Phosphoproteins; D010768:Phosphoserine; D010769:Phosphothreonine; D011433:Propranolol; D054645:Protein Phosphatase 1; D011506:Proteins; C024833:protein phosphatase inhibitor-2; D000097847:Protein Phosphatase Inhibitory Proteins