Properties of p19, a novel cAMP-dependent protein kinase substrate protein purified from bovine brain.
We report the purification from bovine brain and describe some of the properties of a 19-kDa protein, p19, which we have previously shown to undergo hormone-dependent, cAMP-mediated phosphorylation in several peptide hormone-producing tumor cells. The procedure for purifying p19 to apparent homogeneity utilized ammonium sulfate fractionation, sequential chromatography on DEAE-cellulose and phenyl-Sepharose, followed by fast protein liquid chromatography using a Mono Q and, finally, a C8 reverse-phase column. The yield was 0.3-0.5 mg of p19/kg of brain. The molecular weight (Mr = 19,000) and frictional ratio (f/f0 = 1.87) of p19, which were derived from its Stokes radius (33 A) and sedimentation constant (s20,w = 1.4), suggest that the native form of p19 is an asymmetrically shaped monomer. We provide evidence to suggest that p19 is isolated as a mixture of molecular forms consisting of an unphosphorylated form and of three phosphoforms indicative of multisite phosphorylation. These forms cosedimented on sucrose density gradients and coeluted on gel filtration, hydrophobic chromatography, and reverse-phase fast protein liquid chromatography. They were resolved from each other by anion-exchange chromatography. The unphosphorylated form (pI 6.2) was phosphorylated by catalytic subunit of cAMP-dependent protein kinase to a stoichiometry of 0.5 mol of P/mol of p19, thereby giving rise to the three phosphoforms (pI 5.8, pI 5.6, and pI 5.2, respectively). We conclude that p19 is a novel cAMP-dependent protein kinase substrate protein that is present in brain and in peptide hormone-producing tumor cells. Its function remains to be identified.
D000818:Animals; D000837:Anion Exchange Resins; D001923:Brain Chemistry; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D007525:Isoelectric Focusing; D008868:Microtubule Proteins; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000302:isolation & purification* / Q000378:metabolism; D010750:Phosphoproteins / Q000302:isolation & purification* / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D012117:Resins, Synthetic; D050777:Stathmin
D000837:Anion Exchange Resins; D008868:Microtubule Proteins; D009419:Nerve Tissue Proteins; D010750:Phosphoproteins; D012117:Resins, Synthetic; D050777:Stathmin; C042855:Mono Q; D011494:Protein Kinases; D002118:Calcium