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Phosphorylation of Three Proteins in the Plasma Membrane of Y-l Adrenal Cells by a Membrane-Bound Adenosine 3',5'-Monophosphate-Dependent Protein Kinase^*

Worcester Foundation for Experimental Biology Shrewsbury, Massachusetts 01545

Address requests for reprints to: Dr. Peter F. Hall, Worcester Foundation for Experimental Biology, 222 Maple Avenue, Shrewsbury, Massachusetts 01545.

Abstract

Highly purified plasma membranes from Y-1 adrenal tumor cells were incubated with [-³²P]ATP with and without cAMP to determine whether endogenous protein substrates are phosphorylated by a cAMP-dependent protein kinase. Three membrane proteins (mol wt 270,000, 35,000, and 17,000) were phosphorylated without cAMP and, to a greater extent, with the nucleotide (0.05–20 µM). Phosphorylation was rapid (<60 sec), specific for cAMP, and occurred exclusively at serine residues. Two of the three proteins (35,000 and 17,000) were phosphorylated in whole cells under the influence of cAMP when the cells were incubated with [³²P]orthophosphate. The cAMP-dependent protein kinase of these plasma membranes was not extracted by Triton X-100 (0.5% wt/vol) nor by KC1 (0.4 M) but was almost completely extracted by the two agents together. By means of photoactivation of 8-azido-[³²P]cAMP, it was found that both regulatory subunits R₁ and R₁₁ are present in the membranes. To the extent that the second messenger acts only by way of protein kinase enzymes, these changes in the three proteins are likely to be important in the responses of the plasma membranes of adrenal cells to ACTH. (Endocrinology 118: 701–708, 1986)

Footnotes

^* This work has been supported by NIH Grants CA-29497 and AM-32236

Received August 7, 1985.

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