The protein of a new gene,Tctex4, interacts with protein kinase CK2beta subunit and is highly expressed in mouse testis

Casein kinase 2 (CK2) is a ubiquitous, multifunctional eukaryotic serine/threonine kinase that phosphorylates an array of proteins. CK2 is a heterotetramer composed of two catalytic (alpha,alpha(')) and two regulatory (beta) subunits. CK2 plays an essential role in regulatory pathways in cell transformation and proliferation. But the role and function of the individual subunits of CK2, which are not in the holoenzyme, are not yet clear. Northern blot analysis reveals the highest CK2beta activity in mouse testicles and brain. By employing a yeast two-hybrid screen to identify the proteins that interact with CK2beta, we have isolated a cDNA clone encoding a 14-kDa protein with homology to dynein light chains and have designated it as Tctex4. CK2beta interacts specifically with Tctex4 both in a yeast two-hybrid system and in an in vitro interaction assay. Northern blot and in situ hybridization showed that Tctex4 is a novel gene that is expressed in mouse testis.     

Association of the AB and CD-EF domains from rat alpha- and beta-parvalbumin

Association of the parvalbumin AB and CD-EF domains was examined in Hepes-buffered saline, pH 7.4, employing fragments from rat alpha and beta. All of the interactions require Ca(2+). In saturating Ca(2+), the alpha AB/alpha CD-EF (alpha/alpha) complex displays an association constant of (7.6 +/- 0.4) x 10(7) M(-1). Ca(2+)-binding data for a mixture of the alpha fragments are compatible with an identical two-site model, yielding an average binding constant of (8.5 +/- 0.2) x 10(5) M(-1). The beta/beta interaction is significantly weaker, exhibiting an association constant of (3.0 +/- 0.6) x 10(6) M(-1). The Ca(2+)-binding constants for beta/beta are likewise diminished, at (1.0 +/- 0.1) x 10(5) and (2.3 +/- 0.2) x 10(4) M(-1). The magnitude of the apparent DeltaDeltaG(degree)' for Ca(2+) binding by alpha/alpha and beta/beta, at 3.4 kcal/mol, approaches that measured for the intact proteins (3.6 kcal/mol) and is substantially larger than the 1.5 kcal/mol value previously measured for the isolated CD-EF domains. This result suggests that the AB domain can modulate the Ca(2+) affinities of the CD and EF sites. Interestingly, the heterologous alpha/beta complex displays a larger association constant [(6.6 +/- 0.4) x 10(6) M(-1)] than the homologous beta/beta complex and heightened Ca(2+) affinity [binding constants of (1.3 +/- 0.1) x 10(6) and (8.8 +/- 0.2) x 10(4) M(-1)]. By contrast, beta/alpha associates more weakly than alpha/alpha and exhibits sharply reduced affinity for Ca(2+). Thus, the interaction between the beta AB domain and beta CD-EF domain may act to attenuate Ca(2+) affinity in the intact protein.     

Live-cell fluorescence imaging reveals the dynamics of protein kinase CK2 individual subunits

Protein kinase CK2 is a multifunctional enzyme which has long been described as a stable heterotetrameric complex resulting from the association of two catalytic (alpha or alpha') and two regulatory (beta) subunits. To track the spatiotemporal dynamics of CK2 in living cells, we fused its catalytic alpha and regulatory beta subunits with green fluorescent protein (GFP). Both CK2 subunits contain nuclear localization domains that target them independently to the nucleus. Imaging of stable cell lines expressing low levels of GFP-CK2alpha or GFP-CK2beta revealed the existence of CK2 subunit subpopulations exhibiting differential dynamics. Once in the nucleus, they diffuse randomly at different rates. Unlike CK2beta, CK2alpha can shuttle, showing the dynamic nature of the nucleocytoplasmic trafficking of the kinase. When microinjected in the cytoplasm, the isolated CK2 subunits are rapidly translocated into the nucleus, whereas the holoenzyme complex remains in this cell compartment, suggesting an intramolecular masking of the nuclear localization sequences that suppresses nuclear accumulation. However, binding of FGF-2 to the holoenzyme triggers its nuclear translocation. Since the substrate specificity of CK2alpha is dramatically changed by its association with CK2beta, the control of the nucleocytoplasmic distribution of each subunit may represent a unique potential regulatory mechanism for CK2 activity.     

Liprin beta 1, a member of the family of LAR transmembrane tyrosine phosphatase-interacting proteins, is a new target for the metastasis-associated protein S100A4 (Mts1)

Metastasis-associated protein S100A4 (Mts1) induces invasiveness of primary tumors and promotes metastasis. S100A4 belongs to the family of small calcium-binding S100 proteins that are involved in different cellular processes as transducers of calcium signal. S100A4 modulates properties of tumor cells via interaction with its intracellular targets, heavy chain of non-muscle myosin and p53. Here we report identification of a new molecular target of the S100A4 protein, liprin beta1. Liprin beta1 belongs to the family of leukocyte common antigen-related (LAR) transmembrane tyrosine phosphatase-interacting proteins that may regulate LAR protein properties via interaction with another member of the family, liprin alpha1. We showed by the immunoprecipitation analysis that S100A4 interacts specifically with liprin beta1 in vivo. Immunofluorescence staining demonstrated the co-localization of S100A4 and liprin beta1 in the cytoplasm and particularly at the protrusion sites of the plasma membrane. We mapped the S100A4 binding site at the C terminus of the liprin beta1 molecule between amino acid residues 938 and 1005. The S100A4-binding region contains two putative phosphorylation sites by protein kinase C and protein kinase CK2. S100A4-liprin beta1 interaction resulted in the inhibition of liprin beta1 phosphorylation by both kinases in vitro.     

Protein p21(WAF1/CIP1) is phosphorylated by protein kinase CK2 in vitro and interacts with the amino terminal end of the CK2 beta subunit

Protein kinase CK2 is a ubiquitous protein that phosphorylates multiple substrates and is composed of catalytic (alpha, alpha') and regulatory (beta) subunits. Abundant evidence relates CK2 to the regulation of cell division. p21(WAF1/CIP1) is a potent inhibitor of cyclin-dependent kinases and of DNA replication and acts as a key inhibitor of cell cycle progression. In this work we examine the relation between these two important proteins. The interaction between the CK2 beta regulatory subunit of CK2 and p21(WAF1/CIP1) has been confirmed. Using a pull-down assay and fusion constructs of glutathione transferase with fragments of CK2 beta and other mutants, it was possible to define that the N-terminal (1-44) portion of CK2 beta contains a p21(WAF1/CIP1) binding site. CK2 reconstituted from recombinant alpha and beta subunits can phosphorylate p21(WAF1/CIP1) in vitro. This phosphorylation is greatly enhanced by histone H1. p21(WAF1/CIP1) can inhibit the phosphorylation of substrate casein by CK2. This inhibition, however, seems to be due to competition by p21(WAF1/CIP1) as an alternate substrate since in order to observe inhibition it is necessary that the concentration of p21 be of the same order of magnitude as the casein substrate concentration. This competition is not related to the binding of p21(WAF1/CIP1) to CK2 beta because it can also be observed when, in the absence of CK beta, CK alpha is used to phosphorylate casein in the presence of the p21.     

Specific in vivo binding of activator of G protein signalling 1 to the Gbeta1 subunit

Activator of G protein signalling 1 (AGS1) is a Ras-like protein that affects signalling through heterotrimeric G proteins. Previous in vitro studies suggest that AGS1 can bind to G(alpha)-GDP subunits and promote nucleotide exchange, leading to activation of intracellular signalling pathways. This model is consistent with in vivo evidence demonstrating that AGS1 activates both G(alpha)- and G(betagamma)-dependent pathways in the absence of ligand. However, it does not easily explain how AGS1 blocks G(betagamma)-dependent, but not G(alpha)-dependent, signalling following receptor activation. We have used yeast two hybrid analysis and co-immunoprecipitation studies in mammalian cells to demonstrate a direct interaction between AGS1 and the G(beta1) subunit of heterotrimeric G proteins. The interaction is specific for G(beta1) and involves the cationic region of AGS1 and the C-terminal region of G(beta1). Possible implications of this novel interaction for the activity of AGS1 are discussed.     

Competitive inhibition of T3 binding to alpha 1 and beta 1 thyroid hormone receptors by fatty acids.

This study was undertaken to investigate whether fatty acids inhibit the binding of T3 to the alpha 1 and beta 1 form of the thyroid hormone receptor. Fatty acids inhibited the binding of T3 to both receptor proteins isolated from a bacterial expression system. The effectiveness of inhibition depends on the chain length and degree of saturation of the fatty acids. The inhibition of T3 binding to the alpha 1 and beta 1 receptor by oleic acid is competitive in nature; the Ki value was 5.4 10(-6) M for the c-erbA alpha 1 protein and 3.3 10(-6) M for the c-erb beta 1 protein. The findings indicate a direct interaction of fatty acids with T3 receptor proteins.     

Phosphorylation of maize and Arabidopsis HMGB proteins by protein kinase CK2alpha

In plants, a variety of chromatin-associated high mobility group (HMG) proteins belonging to the HMGB family have been identified. We have examined the phosphorylation of the HMGB proteins from the monocotyledonous plant maize and the dicotyledonous plant Arabidopsis by protein kinase CK2alpha. Maize CK2alpha phosphorylates the maize HMGB1 and HMGB2/3 proteins and the Arabidopsis HMGB1, HMGB2/3, and HMGB4 proteins. Maize HMGB4 and HMGB5 and Arabidopsis HMGB5 are not phosphorylated by CK2alpha. Depending on the HMGB protein up to five amino acid residues are phosphorylated in the course of the phosphorylation reaction. The HMGB1 proteins from both plants are markedly more slowly phosphorylated by CK2alpha than the other HMGB substrate proteins, indicating that certain HMGB proteins are clearly preferred substrates for CK2alpha. The rate of the phosphorylation reaction appears to be related to the ease of interaction between CK2alpha and the HMGB proteins, as indicated by chemical cross-linking experiments. MALDI/TOF mass spectrometry analyses demonstrate that the HMGB1 and HMGB2/3 proteins occur in various phosphorylation states in immature maize kernels. Thus, HMGB1 exists as monophosphorylated, double-phosphorylated, triple-phosphorylated, and tetraphosphorylated protein in kernel tissue, and the tetraphosphorylated form is the most abundant version. The observed in vivo phosphorylation states indicate that protein kinase(s) other than CK2alpha contribute(s) to the modification of the plant HMGB proteins. The fact that the HMGB proteins are phosphorylated to various extents reveals that the existence of differentially modified forms increases the number of distinct HMGB protein variants in plant chromatin that may be adapted to certain functions.     

Maize protein kinase CK2: regulation and functionality of three beta regulatory subunits

Biochemical and crystallographic data suggest that, in contrast with other organisms, the active maize protein kinase CK2 might be composed simply of a catalytic polypeptide (CK2alpha), thus lacking CK2beta regulatory subunits. To investigate the existence and functionality of CK2beta regulatory subunits in Zea mays, we have screened a maize cDNA library using different approaches and have isolated three full-length cDNAs encoding CK2beta regulatory subunits (CK2beta-1, CK2beta-2 and CK2beta-3) and a cDNA coding for a novel CK2alpha catalytic subunit, CK2alpha-3. The pattern of expression of all these alpha/beta subunits has been studied in different organs and developmental stages using specific probes for each isoform, and indicates that while CK2alpha subunits are constitutive, CK2beta subunits are expressed differentially during embryo development. The yeast two-hybrid system and pull-down assays have been used to study specific interactions between the different subunits. While CK2alpha subunits are unable to self-associate, preferential interactions between alpha/beta isoforms and beta/beta isoforms can be predicted. Furthermore, we show that maize CK2alpha/beta subunits assemble into a structural tetrameric complex which has very similar properties to those described in other organisms, and that expression of maize CK2beta subunits in yeast allows the rescue of the phenotypic defects associated to the lack of CK2 function, thus demonstrating the functionality of maize CK2beta regulatory subunits.     

Phosphorylation of the regulatory beta-subunit of protein kinase CK2 by checkpoint kinase Chk1: identification of the in vitro CK2beta phosphorylation site

The regulatory beta-subunit of protein kinase CK2 mediates the formation of the CK2 tetrameric form and it has functions independent of CK2 catalytic subunit through interaction with several intracellular proteins. Recently, we have shown that CK2beta associates with the human checkpoint kinase Chk1. In this study, we show that Chk1 specifically phosphorylates in vitro the regulatory beta-subunit of CK2. Chymotryptic peptides and mutational analyses have revealed that CK2beta is phosphorylated at Thr213. Formation of a stable complex between CK2beta and Chk1 is not affected by the modification of Thr213 but it does require the presence of an active Chk1 kinase.     

Mass spectrometry analysis of a protein kinase CK2beta subunit interactome isolated from mouse brain by affinity chromatography

CK2, an acronym derived from the misnomer "casein kinase 2", denotes a ubiquitous and extremely pleiotropic Ser/Thr protein kinase, the holoenzyme of which is composed of two catalytic (alpha and/or alpha') and two noncatalytic beta subunits acting as a docking platform and the multifarious functions of which are still incompletely understood. By combining affinity chromatography and mass spectrometry, we have identified 144 mouse brain proteins that associate with immobilized CK2beta. A large proportion (60%) of the identified proteins had been previously reported to be functionally related to CK2, and a similar proportion have been classified as phosphoproteins with approximately half of these having the features of CK2 targets. A large number of the identified proteins ( approximately 40%) either are nuclear or shuttle between the nucleus and cytoplasm, and the biggest functional classes of CK2beta interactors are committed to protein synthesis and degradation (32 proteins) and RNA/DNA interaction (20 proteins). Also well represented are the categories of cytoskeletal/structural proteins (19), trafficking proteins (17), and signaling proteins (14). The identified proteins are examined in relation to their functions and potential as targets and/or regulators of CK2, disclosing in some cases unanticipated links between this kinase and a variety of biochemical events.     

Eukaryotic initiation factor 2 alpha subunit associates with TGF beta receptors and 14-3-3 epsilon and acts as a modulator of the TGF beta response.

Schistosoma mansoni receptor kinase 1 (SmRK1) is a divergent member of the TGF beta receptor family. Intracellular proteins that associate with these receptors are likely to play an important role in signaling. 14-3-3 epsilon is a previously described cytoplasmic protein, which associates with both SmRK1 and the human type I TGF beta receptor (T beta RI); overexpression of 14-3-3 epsilon leads to enhanced TGF beta-mediated signaling by T beta RI. We now describe the identification of S. mansoni eukaryotic translation initiation factor 2 alpha subunit (eIF2 alpha), through its interaction with SmRK1 in a yeast two-hybrid assay. S. mansoni eIF2 alpha also interacts with human TGF beta receptors. Strongest association was demonstrated with kinase inactive receptors, particularly the type II TGF beta receptor (T beta RII). Both T beta RI and T beta RII phosphorylate eIF2 alpha in vitro, at sites other than the previously described eIF2 alpha phosphorylation sites. EIF2 alpha also modulates signaling by TGF beta receptors; however, in contrast to 14-3-3 epsilon, eIF2 alpha overexpression inhibits the TGF beta-driven response. These data suggest a novel function for eIF2 alpha in the TGF beta signaling pathway. In addition, we have demonstrated an independent interaction between eIF2 alpha and 14-3-3 epsilon. Coexpression of 14-3-3 epsilon with eIF2 alpha leads to the abrogation of the inhibitory effect of eIF2 alpha on TGF beta-mediated signaling. The interaction of these two regulatory proteins with each other and with the TGF beta receptors and their relative expression levels are likely to be important in fine-tuning the regulation of TGF beta signal transduction.     

Active caspase-1 is a regulator of unconventional protein secretion

Mammalian cells export most proteins by the endoplasmic reticulum/Golgi-dependent pathway. However, some proteins are secreted via unconventional, poorly understood mechanisms. The latter include the proinflammatory cytokines interleukin(IL)-1beta, IL-18, and IL-33, which require activation by caspase-1 for biological activity. Caspase-1 itself is activated by innate immune complexes, the inflammasomes. Here we show that secretion of the leaderless proteins proIL-1alpha, caspase-1, and fibroblast growth factor (FGF)-2 depends on caspase-1 activity. Although proIL-1alpha and FGF-2 are not substrates of the protease, we demonstrated their physical interaction. Secretome analysis using iTRAQ proteomics revealed caspase-1-mediated secretion of other leaderless proteins with known or unknown extracellular functions. Strikingly, many of these proteins are involved in inflammation, cytoprotection, or tissue repair. These results provide evidence for an important role of caspase-1 in unconventional protein secretion. By this mechanism, stress-induced activation of caspase-1 directly links inflammation to cytoprotection, cell survival, and regenerative processes.     

The calcium sensor protein visinin-like protein-1 modulates the surface expression and agonist sensitivity of the alpha 4beta 2 nicotinic acetylcholine receptor

The calcium sensor protein visinin-like protein-1 (VILIP-1) was isolated from a brain cDNA yeast two-hybrid library using the large cytoplasmic domain of the alpha4 subunit as a bait. VILIP-1 is a myristoylated calcium sensor protein that contains three functional calcium binding EF-hand motifs. The alpha4 subunit residues 302-339 were found to be essential for the interaction with VILIP-1. VILIP-1 coimmunopurified with detergent-solubilized recombinant alpha4beta2 acetylcholine receptors (AChRs) expressed in tsA201 cells and with native alpha4 AChRs isolated from brain. Coexpression of VILIP-1 with recombinant alpha4beta2 AChRs up-regulated their surface expression levels approximately 2-fold and increased their agonist sensitivity to acetylcholine approximately 3-fold. The modulation of the recombinant alpha4beta2 AChRs by VILIP-1 was attenuated in VILIP-1 mutants that lacked the ability to be myristoylated or to bind calcium. Collectively, these results suggest that VILIP-1 represents a novel modulator of alpha4beta2 AChRs that increases their surface expression levels and agonist sensitivity in response to changes in the intracellular levels of calcium.     

Rhodopsin and the retinal G-protein distinguish among G-protein beta gamma subunit forms

The beta gamma subunits of G-proteins are composed of closely related beta 35 and beta 36 subunits tightly associated with diverse 6-10 kDa gamma subunits. We have developed a reconstitution assay using rhodopsin-catalyzed guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) binding to resolved alpha subunit of the retinal G-protein transducin (Gt alpha) to quantitate the activity of beta gamma proteins. Rhodopsin facilitates the exchange of GTP gamma S for GDP bound to Gt alpha beta gamma with a 60-fold higher apparent affinity than for Gt alpha alone. At limiting rhodopsin, G-protein-derived beta gamma subunits catalytically enhance the rate of GTP gamma S binding to resolved Gt alpha. The isolated beta gamma subunit of retinal G-protein (beta 1, gamma 1 genes) facilitates rhodopsin-catalyzed GTP gamma S exchange on Gt alpha in a concentration-dependent manner (K0.5 = 254 +/- 21 nM). Purified human placental beta 35 gamma, composed of beta 2 gene product and gamma-placenta protein (Evans, T., Fawzi, A., Fraser, E.D., Brown, L.M., and Northup, J.K. (1987) J. Biol. Chem. 262, 176-181), substitutes for Gt beta gamma reconstitution of rhodopsin with Gt alpha. However, human placental beta 35 gamma facilitates rhodopsin-catalyzed GTP gamma S exchange on Gt alpha with a higher apparent affinity than Gt beta gamma (K0.5 = 76 +/- 54 nM). As an alternative assay for these interactions, we have examined pertussis toxin-catalyzed ADP-ribosylation of the Gt alpha subunit which is markedly enhanced in rate by beta gamma subunits. Quantitative analyses of rates of pertussis modification reveal no differences in apparent affinity between Gt beta gamma and human placental beta 35 gamma (K0.5 values of 49 +/- 29 and 70 +/- 24 nM, respectively). Thus, the Gt alpha subunit alone does not distinguish among the beta gamma subunit forms. These results clearly show a high degree of functional homology among the beta 35 and beta 36 subunits of G-proteins for interaction with Gt alpha and rhodopsin, and establish a simple functional assay for the beta gamma subunits of G-proteins. Our data also suggest a specificity of recognition of beta gamma subunit forms which is dependent both on Gt alpha and rhodopsin. These results may indicate that the recently uncovered diversity in the expression of beta gamma subunit forms may complement the diversity of G alpha subunits in providing for specific receptor recognition of G-proteins.     

Alternatively spliced FGFR-1 isoform signaling differentially modulates endothelial cell responses to peroxynitrite

Mounting experimental evidence has suggested that the trophic environment of cells in culture is an important determinant of their vulnerability to the cytotoxic effects of reactive oxidants such as peroxynitrite (ONOO(-)). However, acidic fibroblast growth factor (FGF-1)-induced signaling renders some cells more sensitive and others resistant to the cytotoxic effects of ONOO(-). To determine whether alternatively spliced fibroblast growth factor receptor (FGFR-1) isoforms are responsible for this differential response, we have stably transfected FGFR-negative rat brain-derived resistant vessel endothelial cells (RVEC) with human cDNA sequences encoding either FGFR-1 alpha or FGFR-1 beta. FGF-1 treatment of RVEC(R-1 alpha) transfectants enhanced ONOO(-)-mediated cell death in a manner dependent upon FGFR-1 tyrosine kinase, MEK/Erk 1/2 kinase, and p38 MAP kinase activities and independent of Src-family kinase (SFK) activity. FGF-1 treatment of RVEC(R-1 beta) transfectants inhibited the cytotoxic effects of ONOO(-) in a manner dependent upon FGFR-1 tyrosine kinase, MEK/Erk 1/2 kinase, and SFK activities and independent of p38 MAP kinase activity. FGF-1-induced preactivation of both FGFR-1 tyrosine and Erk 1/2 kinases was detected in both RVEC(R-1 alpha) and RVEC(R-1 beta) transfectants. FGF-1-induced preactivation of p38 MAPK was restricted to RVEC(R-1 alpha) transfectants, whereas, ligand-induced preactivation of SFK was limited to RVEC(R-1 beta) transfectants. Collectively, these results both reemphasize the role of extracellular trophic factors and their receptor-mediated signaling pathways during cellular responses to oxidant stress and provide a first indication that the alternatively spliced FGFR-1 isoforms induce differential signal transduction pathways.