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Volume 271, Number 23, Issue of June 7, 1996 pp. 13308-13316
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Role of Phosphorylation on DNA Binding and Transcriptional Functions of Human Progesterone Receptors

(Received for publication, December 6, 1995, and in revised form, March 29, 1996)

Glenn S. Takimoto , Alicia Rudie Hovland ^§ , Diane M. Tasset , Mary Y. Melville , Lin Tung and Kathryn B. Horwitz ^§''

From the Departments of  Medicine and ^'' Pathology and the ^§ Molecular Biology Program, Division of Endocrinology, Metabolism and Diabetes, University of Colorado Health Sciences Center, Denver, Colorado 80262

To study the function of human progesterone receptor (hPR) phosphorylation, we have tested four sets of serine to alanine substitution mutants: 10 serine clusters, located in regions common to both hPR isoforms (the M-series mutants) were mutated in A-receptors and B-receptors; 6 serine clusters located in the B-upstream segment (BUS; the B-series mutants) were mutated individually and collectively and cloned into B-receptors and into BUS-DBD-NLS, a constitutive transactivator, in which the AF3 function of BUS is fused to the DNA binding domain (DBD) and nuclear localization signal (NLS) of hPR. Transcription by most of the M-series mutants resembles that of wild-type A- or B-receptors. Mutation of 3 sites, Ser¹⁹⁰ at the N terminus of A-receptors, a cluster of serines just upstream of the DBD, or Ser⁶⁷⁶ in the hinge region, inhibits transcription by 20-50% depending on cell or promoter context. These sites lie outside the AF1 activation function. M-series mutants are substrates for a hormone-dependent phosphorylation step, and they all bind well to DNA. Progressive mutation of the B-series clusters leads to the gradual dephosphorylation of BUS, but only the 6-site mutant, involving 10 serine residues, is completely dephosphorylated. These data suggest that in BUS alternate serines are phosphorylated or dephosphorylated at any time. However, even when BUS is completely dephosphorylated, both BUS-DBD-NLS and full-length B-receptors remain strong transactivators. Mutant B-receptors also do not acquire the dominant negative properties of A-receptors, and they retain the ability to activate transcription in synergy with 8-Br-cAMP and antiprogestins. We conclude that phosphorylation has subtle effects on the complex transcriptional repertoire that distinguishes the two hPR isoforms and does not influence transactivation mediated by AF1 or AF3, but subserves other functions.

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