ABSTRACT
Rat osteo-testicular protein tyrosine phosphatase (OST-PTP), expressed in osteoblasts and testis, is a receptor-like transmembrane protein with two tandemly repeated phosphatase domains in the cytoplasmic region. In this report, we show that the first domain (CD1) is enzymatically active and appears to be influenced by the catalytically inactive second domain (CD2). The activity of CD1 is specific to phosphorylated tyrosine. Full-length OST-PTP protein expressed in COS cells has a molecular mass of approximately 185 kDa, and immunoprecipitates of this protein using OST-PTP-specific antisera show strong tyrosine phosphatase activity. Expression of OST-PTP mRNA in primary rat calvarial osteoblasts is temporally regulated, and peak expression is found at approximately day 15, which correlated well with the appearance of OST-PTP protein and its associated tyrosine phosphatase activity. Treatment of osteoblasts in culture with antisense oligonucleotides directed against the 5' untranslated region of OST-PTP results in abrogation of differentiation, confirming the functional importance of OST-PTP expression in osteoblast development.
Subject(s)
Osteoblasts/enzymology , Protein Tyrosine Phosphatases/chemistry , Testis/enzymology , Animals , COS Cells , Cell Differentiation/drug effects , Cell Differentiation/genetics , Cells, Cultured , Chlorocebus aethiops , Enzyme Activation/genetics , Enzyme Inhibitors/pharmacology , Fetus , Growth Inhibitors/genetics , Growth Inhibitors/pharmacology , Male , Mutagenesis, Site-Directed , Oligonucleotides, Antisense/pharmacology , Osteoblasts/cytology , Osteoblasts/drug effects , Protein Structure, Tertiary/genetics , Protein Tyrosine Phosphatases/antagonists & inhibitors , Protein Tyrosine Phosphatases/biosynthesis , Protein Tyrosine Phosphatases/genetics , RNA, Messenger/biosynthesis , Rats , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , TransfectionABSTRACT
Estrogen receptors ERalpha and ERbeta are members of the family of nuclear hormone receptors and act as ligand-inducible transcriptional factors, which regulate the expression of target genes on binding to cognate response elements. We report here the characterization of steroid receptor coactivator-3 (SRC-3), a coactivator of nuclear receptor transcription that is a member of a family of steroid receptor coactivators that includes SRC-1 and transcription intermediate factor-2. SRC-3 enhanced ERalpha and progesterone receptor-stimulated gene transcription in a ligand-dependent manner, but stimulation of ERbeta-mediated transcription was not observed. Protein-protein interaction assays, including real-time interaction analyses with BIAcore, demonstrated that the affinity of the ERalpha interaction with SRC-3 was much higher than that observed for the ERbeta interaction with SRC-3. Mutational analysis suggests a potential interplay between the transactivation function-1 and -2 domains of ERalpha and SRC-3. Furthermore, an intrinsic transactivation function was observed in the C-terminal half of SRC-3. Finally, SRC-3 was differentially expressed in various tissues and, among several tumor cells examined, was most abundant in the nuclear fraction of MCF-7 breast cancer cells. Therefore, SRC-3, a third member of a family of steroid receptor coactivators, has a distinct tissue distribution and intriguing selectivity between ERalpha and ERbeta.
