A PIT-1 PHOSPHORYLATION MUTANT CAN MEDIATE BOTH BASAL AND INDUCED PROLACTIN AND GROWTH-HORMONE PROMOTER ACTIVITY

The transcription factor Pit-1 has been shown to be important for both the developmental and homeostatic regulation of expression of the PRL and GH genes in pituitary cells. However, little is known about possible covalent modifications in Pit-1 that might mediate its transactivational properties. Previous studies showing that Pit-1 is a phosphorylation substrate for either protein kinase A or C, or their cellular inducers, led us to investigate whether phosphorylation of Pit-1 is required for its function in either basal or induced cellular activity of either the PRL or GH promoters. The transactivational properties of wild type Pit-1 were compared with those of Pit-1(A3), mutated in the three known phosphorylation sites. At saturating levels of Pit-1 expression vectors, activation of transient basal expression in HeLa cells of constructs (-1957)PRL-CAT or (-244)GH-CAT by RSV-Pit-1(A3) was, respectively, about 50% and 65% as strong as by RSV-Pit-1. Hence, phosphorylation at the sites mutated in Pit-1(A3) is not critically required for basal transactivation of either promoter but may modulate this activity. RSV-Pit-1 and RSV-Pit-1(A3) were equally effective in mediating estrogen receptor stimulation of (-1957)PRL-CAT expression in HeLa cells, thus revealing no phosphorylation requirement for the prerequisite for Pit-1 in estrogen receptor action on the PRL estrogen response element. A possible functional requirement for Pit-1 phosphorylation was further investigated by examining the abilities of forskolin and 12-O-tetradecanoyl phorbol-13-acetate, cellular inducers of, respectively, protein kinases A and C, to stimulate transient expression of (-187)PRL-CAT in AtT20 pituitary cells transfected with either RSV-Pit-1 or RSV-Pit-1(A3). Cells expressing either Pit-1 or Pit-1(AB) yielded indistinguishable responses to either forskolin or TPA, implying that the ability of the corresponding kinases to stimulate PRL promoter activity does not depend upon the phosphorylation state of Pit-1. These results imply that neither basal expression of either the PRL or GH genes, nor induction of the PRL gene by the agents examined, is critically dependent upon phosphorylation of Pit-1 at any of the known sites.