The serum-and glucocorticoid-inducible kinase (sgk) is a novel serine/threonine protein kinase that is transcriptionally regulated in rat mammary tumor cells by serum under proliferative conditions or by glucocorticoids that induce a G 1 cell cycle arrest. Our results establish that the subcellular distribution of Sgk is under stringent cell cycle and hormonal control. Sgk is localized to the perinuclear or cytoplasmic compartment as a 50-kDa hypophosphorylated protein in cells arrested in G 1 by treatment with the synthetic glucocorticoid dexamethasone. In serum-stimulated cells, Sgk was transiently hyperphosphorylated and resided in the nucleus. Laser scanning cytometry, which monitors Sgk localization and DNA content in individual mammary tumor cells of an asynchronously growing population, revealed that Sgk actively shuttles between the nucleus (in S and G 2 /M) and the cytoplasm (in G 1 ) in synchrony with the cell cycle. In cells synchronously released from the G 1 /S boundary, Sgk localized to the nucleus during progression through S phase. The forced retention of exogenous Sgk in either the cytoplasmic compartment, using a wild type sgk gene, or the nucleus, using a nuclear localization signal-containing sgk gene (NLS-Sgk), suppressed the growth and DNA synthesis of serumstimulated cells. Thus, our study implicates the nuclearcytoplasmic shuttling of sgk as a requirement for cell cycle progression and represents a novel convergence point of anti-proliferative and proliferative signaling in mammary tumor cells.A dynamic balance of steroid hormones, protein growth factors, and other environmental cues coordinately regulates an intricate network of intracellular processes that stringently control mammalian cell proliferation (1-5). A large body of literature has characterized the individual cellular events activated by either steroid hormones (6 -10) or by protein hormones and growth factors (3,(11)(12)(13)(14)(15), which trigger the two principal signal transduction pathways that eukaryotic cells employ to respond to external stimuli. To understand the functional connections between the transcriptional events regulated by nuclear steroid receptors (7,8,10,16, 17) and the cascades of phosphorylation-dephosphorylation reactions mediated by the cell-surface growth factor receptors (3,11,13,15,18), a crucial issue was to define the key steps at which these signal transduction pathways converge. There are a variety of potential mechanisms of cross-talk between growth factor and steroid-responsive pathways (5, 19). These regulatory steps include steroid-mediated changes in the expression of growth factors, their cognate receptors, and components of phosphorylation and dephosphorylation cascades (20 -29). In other cellular contexts, the phosphorylation of steroid receptors can alter their function and target gene specificity (30, 31). The regulation of cell signaling events in the nucleus for the coordinate control of target genes allows cells to respond to external stimuli in a physiologically appropriate manne...

sgk is a novel member of the serine/threonine protein kinase gene family that is transcriptionally regulated by serum and glucocorticoids in mammary epithelial cells. To functionally determine if the sgk promoter is regulated by the p53 tumor suppressor protein in mammary cells, a series of sgk promoter fragments with 5 -deletions were linked to the bacterial chloramphenicol acetyltransferase gene (sgk-CAT) and transiently cotransfected into nontumorigenic NMuMG or transformed Con8Hd6 mammary epithelial cells with p53 expression plasmids. Wild-type p53, but not mutant p53, strongly stimulated sgk promoter activity in both mammary epithelial cell lines. These effects were mediated by specific regions within the sgk promoter containing p53 DNA-binding sites. The sgk p53 sequence at ؊1380 to ؊1345 (site IV) was sufficient to confer p53-dependent transactivation to a heterologous promoter, and p53 was capable of binding to this sequence in vitro as assessed by gel shift analysis. In the nontumorigenic NMuMG epithelial cell line, cotransfection of wild-type p53 strongly stimulated the activities of both the sgk promoter and the well characterized p53-responsive p21/ Waf1 promoter, whereas in Rat-2 fibroblasts, wild-type p53 repressed the basal activities of both promoters, revealing that sgk and p21/Waf1 are similarly regulated in a cell type-specific manner. Taken together, these results demonstrate that sgk is a new transcriptional target of p53 in mammary epithelial cells and represent the first example of a hormone-regulated protein kinase gene with a functionally defined p53 promoter recognition element.An intricate network of protein kinases and phosphatases propagates various extracellular growth and differentiation signals from the plasma membrane into the nucleus, leading to changes in the phosphorylation status and the function of discrete sets of transcription factors. The catalytic activities of most protein kinases are regulated by specific interactions with regulatory proteins (1-3) and/or by phosphorylation (4, 5). Recent studies have uncovered a newly emerging subfamily of serine/threonine protein kinase genes, including snk, sgk, plk, and fnk, that are predominantly regulated at the transcriptional level by hormone-and/or mitogen-induced pathways (6 -13). Our previous studies have identified the sgk (serum-and glucocorticoid-inducible protein kinase) serine/threonine protein kinase gene, which is transcriptionally regulated by serum and/or glucocorticoids in mammary epithelial cells and Rat-2 fibroblasts (12, 13), as the second member of this subfamily of transcriptionally regulated protein kinase genes. sgk encodes a 49-kDa putative protein kinase that shares 45-55% homology with the catalytic domain of protein kinase C, the cAMP-dependent protein kinase A, the rac protein kinases, and the ribosomal protein S6 kinase (13). We have documented that sgk transcripts are expressed in a variety of adult rat tissues, with the highest expression in the thymus, lung, and ovary and detectable levels in the ma...

sgk is a novel member of the serine/threonine protein kinase family that is transcriptionally regulated by serum and glucocorticoids in Rat2 fibroblasts and in mammary epithelial cells. 5'-Deletion analysis of the sgk promoter, using a series of sgk-CAT. (chloramphenicol acetyltransferase) chimeric reporter gene plasmids, defined a glucocorticoid-responsive region that contains a glucocorticoid response element (sgkGRE) between -1000 and -975 bp. The sgkGRE is specifically bound by glucocorticoid receptors and is sufficient to confer glucocorticoid responsiveness to a heterologous promoter in several cell lines. Strikingly, cotransfection of either the murine or human wild type p53, but not a mutant p53, repressed the dexamethasone-stimulated transactivation of reporter plasmids containing either the sgkGRE or a consensus GRE. Gel shift analysis revealed that in vitro synthesized p53 prevented binding of the glucocorticoid receptor both to the sgkGRE as well as to a consensus GRE. The p53-mediated repression of dexamethasone-induced sgkGRE activity required both the DNA binding and transactivation functions of the p53 protein. Activation of endogenous p53, by exposure to UV light, repressed the glucocorticoid receptor transactivation of a consensus GRE-CAT reporter plasmid in transfected cells. Conversely, activated glucocorticoid receptors suppressed the transactivation function of p53, while transrepression by p53 was largely unaffected. The presented data demonstrate that sgk is a primary glucocorticoid-responsive protein kinase gene that implicates a new pathway of cross-talk between steroid receptor signaling and cellular phosphorylation cascades. In addition, our study provides the first evidence of mutual interference of transactivation functions of p53 and the glucocorticoid receptor, possibly through their direct interaction.