ARGININE-VASOPRESSIN (AVP) CAUSES THE REVERSIBLE PHOSPHORYLATION OF THE MYRISTOYLATED ALANINE-RICH C-KINASE SUBSTRATE (MARCKS) PROTEIN IN THE OVINE ANTERIOR-PITUITARY - EVIDENCE THAT MARCKS PHOSPHORYLATION IS ASSOCIATED WITH ADRENOCORTICOTROPIN (ACTH) SECRETION (VOL 101, PG 247,1994)

We have recently shown that AVP causes a protein kinase C (PKC)-depend ent increase in ACTH release and biosynthesis in ovine anterior pituit ary cells. In these cells, AVP also causes the translocation of PKC fr om the cytosol to the cell membrane which is maximal at 5 min, but the intracellular events distal to protein kinase C activation that under lie ACTH secretion have not been well characterized to date. Since the MARCKS protein has been implicated in neurosecretion and is phosphory lated by PKC in synaptosomes, studies were carried out to determine wh ether AVP might cause MARCKS phosphorylation in the ovine anterior pit uitary, and to determine whether this phenomenon might be temporally c orrelated with PKC translocation and the release of ACTH. When cytosol ic fractions of rat brain, ovine anterior pituitary, and cultured ovin e anterior pituitary cells were incubated with purified PKC, several p roteins were phosphorylated including those in the region of 83-85 kDa . After precipitation of the proteins with 40% acetic acid, the 83-85 kDa phosphoproteins were selectively recovered in the acid soluble pha se. Phosphopeptide maps of either the 83 or 85 kDa proteins were gener ated with Staphylococcus aureus V8 protease and revealed 13 and 9 kDa phosphopeptides, which are characteristic of the authentic MARCKS prot ein. An identical phosphopeptide map was also obtained when the MARCKS protein was selectively extracted from intact P-32-labeled anterior p ituitary cells. MARCKS phosphorylation was markedly increased when ovi ne anterior pituitary cells were exposed to 1 mu M phorbol 12-myristat e 13-acetate (PMA). When the cells were exposed to 1 mu M AVP, MARCKS phosphorylation increased at 15 s and reached the maximal plateau valu e at 30 s. MARCKS phosphorylation then started to diminish at 2 min, a nd baseline levels were attained by 10 min. In the same cells, AVP sti mulated ACTH release in a biphasic manner - during the first 30 s, the re resulted a rapid burst of ACTH secretion that was followed by a slo wer, but sustained rate of secretion. We conclude that: (1) AVP causes a rapid, and reversible, phosphorylation of the MARCKS protein in the ovine anterior pituitary; (2) since the AVP-induced increase in MARCK S phosphorylation occurs much earlier in these cells than does PKC tra ns-location, MARCKS phosphorylation may provide a more sensitive index of the onset of PKC activation than the translocation assay; (3) the close temporal association between MARCKS phosphorylation and the rapi d early release of ACTH suggests that MARCKS phosphorylation may be in volved in the initial intracellular events that underly exocytosis of the hormone.