Phosphorylation of Pro - ACTH / Endorphin - derived Peptides 4909

The possible occurrence of phosphorylation of proadrenocorticotropin(ACTH)/endorphin-derived peptides in the rat was examined by incubating dissociated anterior and intermediate pituitary cells with a %amino acid plus 32Pi. In both lobes of the pituitary newly synthesized pro-ACTH/endorphin was labeled with 32P. In the intermediate pituitary, corticotropin-like intermediate lobe peptide (CLIP) and glycosylated CLIP were the major phosphorylated product peptides; phosphorylated ACTH and glycosylated ACTH were also detected. In the anterior pituitary ACTH and glycosylated ACTH were the major phosphorylated product peptides; phosphorylated ACTH biosynthetic intermediate was also detected. Glycosylated and nonglycosylated ACTH and CLIP were phosphorylated to approximately the same extent. In both lobes of the pituitary, 16,000 daltons fragment-related peptides were phosphorylated, but to an approximately 10-fold lesser extent than CLIP or ACTH. No detectable phosphorylation of 8-lipotropin, y-lipotropin, or 8-endorphin occurred. Based on isoelectric focusing of double labeled samples, approximately two-thirds of the CLIP in the rat intermediate lobe gets phosphorylated; based on COOH-terminal ACTH immunoassay a similar fraction of the CLIP in fresh intermediate lobe extracts is phosphorylated. Alkaline phosphatase treatment converts phosphorylated CLIP into CLIP. In anterior pituitary extracts roughly half of the ACTH is phosphorylated. In the mouse intermediate pituitary the phosphorylation pattern is qualitatively similar to that described for rat intermediate pituitary but only approximately a fifth of the CLIP is phosphorylated; no phosphorylation of 8-lipotropin, y-lipotropin, or 8-endorphin was observed. In bovine intermediate pituitary no phosphorylation of CLIP was detected in double label incubations or in tissue extracts. The serine residue phosphorylated in rat and mouse ACTH and CLIP (Ser’l) could be phosphorylated by a protein kinase with specificity similar to that of “physiological casein kinases,” which require the sequence Ser-X-Acidic.