In this study, we show that the ETS transcription factor ER81 directly binds to and activates the promoter of the matrix metalloproteinase gene, MMP-1. Further, the oncoprotein HER2/Neu synergizes with ER81 to stimulate MMP-1 transcription. The activation of ER81 by HER2/Neu is mediated by MAP kinases, which phosphorylate ER81 in its N-terminal activation domain. Four respective phosphorylation sites have been identified. Blocking phosphorylation at these sites decreases ER81 transcriptional activity, which can be further diminished by abolishment of phosphorylation at two non-MAP kinase sites. Altogether, our results reveal mechanisms of how phosphorylation of ER81 regulates the expression of target genes such as MMP-1, which may be important for many physiological processes from embryogenesis to adulthood as well as for tumor metastasis.

The catalytic subunits of protein kinase CK2, CK2␣ and CK2␣, are closely related to each other but exhibit functional specialization. To test the hypothesis that specific functions of CK2␣ and CK2␣ are mediated by specific interaction partners, we used the yeast twohybrid system to identify CK2␣-or CK2␣-binding proteins. We report the identification and characterization of a novel CK2-interacting protein, designated CKIP-1, that interacts with CK2␣, but not CK2␣, in the yeast two-hybrid system. CKIP-1 also interacts with CK2␣ in vitro and is co-immunoprecipitated from cell extracts with epitope-tagged CK2␣ and an enhanced green fluorescent protein fusion protein encoding CKIP-1 (i.e. EGFP-CKIP-1) when they are co-expressed. CK2 activity is detected in anti-CKIP-1 immunoprecipitates performed with extracts from non-transfected cells indicating that CKIP-1 and CK2 interact under physiological conditions. The CKIP-1 cDNA is broadly expressed and encodes a protein with a predicted molecular weight of 46,000. EGFP-CKIP-1 is localized within the nucleus and at the plasma membrane. The plasma membrane localization is dependent on the presence of an amino-terminal pleckstrin homology domain. We postulate that CKIP-1 is a non-enzymatic regulator of one isoform of CK2 (i.e. CK2␣) with a potential role in targeting CK2␣ to a particular cellular location. Protein kinase CK2 (CK2)1 is an essential, highly conserved, protein serine/threonine kinase present in all eukaryotic cells (reviewed in Refs. 1-6). CK2 has been reported to phosphorylate a broad range of cellular proteins located in a variety of cellular compartments (mainly the nucleus and cytoplasm) and is involved in important cellular processes such as transcription, translation, morphogenesis, and cell cycle progression (reviewed in Refs. 1-7). These observations support an important role for CK2 in a variety of cellular functions; however, its specific roles and mode of regulation in cells remain poorly understood. Moreover, these results suggest that CK2 is involved in a complex array of interactions with a wide selection of cellular proteins that are present in a broad variety of cellular locations.CK2 is a tetrameric protein comprised of two regulatory subunits (CK2␤) and two catalytic subunits (CK2␣ and/or CK2␣Ј). CK2␣ and CK2␣Ј are the products of separate genes, and their amino acid sequences are highly conserved between higher eukaryotes (reviewed in Ref. 7). In fact, in mammals and birds, CK2␣ and CK2␣Ј exhibit greater than 90% identity over their 330 amino-terminal amino acids (7). This aminoterminal sequence identity is in stark contrast to the unrelated carboxyl-terminal sequences of CK2␣ and CK2␣Ј that exhibit no obvious similarity (8 -10). This sequence divergence between the carboxyl-terminal domains of CK2␣ and CK2␣Ј suggests that important functional differences that exist between the two different catalytic isozymes result from these unique sequences (11).Previous studies have failed to demonstrate significant catalytic differences between CK2␣ and...

Cardiac gap junction (GJ) channels, composed of connexins, allow electrical and metabolic couplings between cardiomyocytes, properties important for coordinated action of the heart as well as tissue homeostasis and control of growth and differentiation. Fibroblast growth factor-2 (FGF-2) is an endogenous growth-promoting protein, believed to participate in the short- and long-term responses of the heart to injury. We have examined short-term effects of FGF-2 on cardiac myocyte GJ-mediated metabolic coupling, using cultures of neonatal rat cardiomyocytes. FGF-2 decreased coupling between cardiomyocytes assessed by scrape dye loading as well as microinjection and dye transfer within 30 minutes of administration. Genistein blocked the effects of FGF-2. To determine the mechanism, we next assessed the effect of FGF-2 on expression, distribution, and phosphorylation of connexin43 (Cx43), which is a major cardiomyocyte connexin. FGF-2 did not affect Cx43 mRNA or protein accumulation and synthesis, and it did not change Cx43 localization at sites of intercellular contact as assessed by immunostaining with a polyclonal anti-Cx43 antibody raised against a synthetic peptide containing residues 346 to 363 of Cx43. FGF-2, however, decreased staining intensity at sites of intermyocyte contact when a monoclonal anti-Cx43 antibody was used, suggesting a localized masking of epitope(s) recognized by the monoclonal but not the polyclonal antibody. These epitopes appear to reside within residues 261 to 270 of Cx43, as indicated by full quenching of monoclonal antibody staining with synthetic peptides. In addition, FGF-2 induced a more than twofold increase in Cx43 phosphorylation. Phosphoamino acid analysis indicated increased phosphorylation of Cx43 on serine residues. Although tyrosine phosphorylation of Cx43 was not detected in either treated or control cells, a fraction of Cx43 was immunoprecipitated with anti-phosphotyrosine-specific antibodies in FGF-2-treated myocytes, suggesting interaction (and hence coprecipitation) with phosphotyrosine-containing protein(s). In conclusion, we have identified Cx43 and intercellular communication as targets of FGF-2-triggered and tyrosine phosphorylation-dependent signal transduction in cardiac myocytes. It is suggested that phosphorylation of Cx43 on serine induced by FGF-2 contributes to decreased metabolic coupling between cardiomyocytes.

. In the present study, we have demonstrated that a glutathione S-transferase fusion protein encoding the C-terminal 126 amino acids of the ␣ subunit is phosphorylated by p34 cdc2 at the same sites as intact casein kinase II, indicating that the mitotic phosphorylation sites are localized within the C-terminal domain of ␣. Four residues within this domain, Thr-344, Thr-360, Ser-362, and Ser-370, conform to the minimal consensus sequence for p34 cdc2 phosphorylation. Synthetic peptides corresponding to regions of ␣ that contain each of these residues are phosphorylated by p34 cdc2 at these sites. Furthermore, alterations in the phosphorylation of the glutathione S-transferase proteins encoding the C-terminal domain of ␣ are observed when any of the four residues are mutated to alanine. When all four residues are mutated to alanine, the fusion protein is no longer phosphorylated by p34 cdc2 at any of the sites that are phosphorylated in mitotic cells. These results indicate that Thr-344, Thr-360, Ser-362, and Ser-370 are the sites on the ␣ subunit of casein kinase II that are phosphorylated in mitotic cells.Biochemical and genetic studies have demonstrated that the p34 cdc2 protein kinase is an indispensable regulator of events leading to the division of eukaryotic cells (for reviews see Refs. 1-4). To ensure that the division of cells is very precisely regulated, the activity of this protein serine/threonine kinase is exquisitely controlled through its interactions with regulatory cyclins and through phosphorylation of p34 cdc2 itself. p34 cdc2 is defined as a cyclin-dependent kinase since it is inactive unless it is associated with a regulatory cyclin subunit. Furthermore, p34 cdc2 is inhibited by phosphorylation of Tyr-15 and/or Thr-14 (5, 6) but requires phosphorylation of Thr-161 to be activated (7,8). CAK (the p34 cdc2 activating kinase) is responsible for phosphorylation of Thr-161 (9 -11), while the phosphorylation state of Thr-14 and/or Tyr-15 is at least in part controlled by the relative activities of the Wee1 protein kinase (12, 13) and cdc25 protein phosphatase (14,15).Concomitant with the activation of p34 cdc2 at the G 2 -M transition of eukaryotic cells is a massive burst of protein phosphorylation. Many of the events that are associated with entry into mitosis including nuclear envelope breakdown, transcriptional termination, nucleolar disassembly, cytoskeletal reorganization, and chromosome condensation appear to be associated with protein phosphorylation. While it is evident that p34 cdc2 directly phosphorylates a number of proteins at the G 2 -M transition, there are also indications that p34 cdc2 could indirectly regulate phosphorylation events through its phosphorylation of other protein kinases (16 -23).One protein serine/threonine kinase that could be regulated by p34 cdc2 is casein kinase II (CKII), 1 which has been shown to be dramatically phosphorylated in mitotic cells (19 -21). CKII is a tetrameric enzyme composed of two catalytic (␣ and/or ␣Ј-subunits) and two additional subunits (␤ s...