SELECTIVE PHOSPHORYLATION OF A NUCLEAR-ENVELOPE POLYPEPTIDE BY AN ENDOGENOUS PROTEIN-KINASE

Incubation of highly purified nuclear envelope with [γ-32P]ATP resulted in the selective phosphorylation of a major membrane polypeptide (Mr 68 000) by an endogenous protein kinase. Phosphorylation of this major polypeptide decreased drastically upon perturbation with low concentrations of Triton X-100 or sodium deoxycholate. Similar results were obtained when the nuclear envelope was sonicated vigorously prior to incubation with [γ-32P]ATP. No stimulation of 32P incorporation was noted in the presence of either cAMP or cGMP. Magnesium ion was required for maximal phosphorylation of the 68 000 molecular weight polypeptide; however, Mn2+ was approximately 40% as effective as Mg2+. No phosphorylation occurred in the presence of Ca2+ or Cu2+. Optimal phosphorylation was obtained between pH 6 and 9. Studies with the membrane matrix of the endoplasmic reticulum revealed the complete absence of the readily labeled 68 000 molecular weight polypeptide found in the nuclear envelope. Phosphorylation of the microsomal membrane was less specific as evidenced by the incorporation of 32P into at least three proteins, the most prominent having molecular weights of 54000 and 49000. Both membranes yielded O-phosphoserine and O-phosphothreonine in a ratio of 3:1 after acid hydrolysis. Partially purified preparations of pore complexes derived from nuclear envelope showed an enrichment of the 68 000 molecular weight protein, suggesting the possible involvement of this highly selective kinase reaction in the exchange of solutes between the nucleus and cytoplasm. © 1979, American Chemical Society. All rights reserved.