Cyclic nucleotide-independent phosphorylation of vitellin by casein kinase II purified from Rhodnius prolixus oocytes

In this study we show that Vitellin (VT) phosphorylation in chorionated oocytes of Rhodnius prolixus is completely inhibited by heparin (10 microg/ml), a classical casein kinase II (CK II) inhibitor. VT phosphorylation is not affected by modulators of cyclic nucleotide-dependent protein kinases such as c-AMP (10 microM), H-8 (1 microM) and H-89 (0.1 microM). We have obtained a 3000-fold VT-free enriched preparation of CK II. Autophosphorylation of this enzyme preparation in the presence of (32)P-ATP demonstrated that it lacks any endogenous substrates. Rhodnius CK II is strongly inhibited by heparin (Ki = 9 nM) and uses ATP (Km = 36 microM) or GTP (Km = 86 microM) as phosphate donors. Incubation of VT with purified Rhodnius CK II and (32)P-ATP led to the incorporation of 2 mols of phosphate/mol VT. However, the total number of phosphorylation sites available can be altered by previous incubation of VT with alkaline phosphatase. These data show that an insect yolk protein contain phosphorylation sites for a cyclic nucleotide-independent protein kinase such as CK II.     

CK2 controls multiple protein kinases by phosphorylating a kinase-targeting molecular chaperone, Cdc37

Cdc37 is a kinase-associated molecular chaperone whose function in concert with Hsp90 is essential for many signaling protein kinases. Here, we report that mammalian Cdc37 is a pivotal substrate of CK2 (casein kinase II). Purified Cdc37 was phosphorylated in vitro on a conserved serine residue, Ser13, by CK2. Moreover, Ser13 was the unique phosphorylation site of Cdc37 in vivo. Crucially, the CK2 phosphorylation of Cdc37 on Ser13 was essential for the optimal binding activity of Cdc37 toward various kinases examined, including Raf1, Akt, Aurora-B, Cdk4, Src, MOK, MAK, and MRK. In addition, nonphosphorylatable mutants of Cdc37 significantly suppressed the association of Hsp90 with protein kinases, while the Hsp90-binding activity of the mutants was unchanged. The treatment of cells with a specific CK2 inhibitor suppressed the phosphorylation of Cdc37 in vivo and reduced the levels of Cdc37 target kinases. These results unveil a regulatory mechanism of Cdc37, identify a novel molecular link between CK2 and many crucial protein kinases via Cdc37, and reveal the molecular basis for the ability of CK2 to regulate pleiotropic cellular functions.     

Unbiased functional proteomics strategy for protein kinase inhibitor validation and identification of bona fide protein kinase substrates: application to identification of EEF1D as a substrate for CK2

Protein kinases have emerged as attractive targets for treatment of several diseases prompting large-scale phosphoproteomics studies to elucidate their cellular actions and the design of novel inhibitory compounds. Current limitations include extensive reliance on consensus predictions to derive kinase-substrate relationships from phosphoproteomics data and incomplete experimental validation of inhibitors. To overcome these limitations in the case of protein kinase CK2, we employed functional proteomics and chemical genetics to enable identification of physiological CK2 substrates and validation of CK2 inhibitors including TBB and derivatives. By 2D electrophoresis and mass spectrometry, we identified the translational elongation factor EEF1D as a protein exhibiting CK2 inhibitor-dependent decreases in phosphorylation in (32)P-labeled HeLa cells. Direct phosphorylation of EEF1D by CK2 was shown by performing CK2 assays with EEF1D -FLAG from HeLa cells. Dramatic increases in EEF1D phosphorylation following λ-phosphatase treatment and phospho- EEF1D antibody recognizing EEF1D pS162 indicated phosphorylation at the CK2 site in cells. Furthermore, phosphorylation of EEF1D in the presence of TBB or TBBz is restored using CK2 inhibitor-resistant mutants. Collectively, our results demonstrate that EEF1D is a bona fide physiological CK2 substrate for CK2 phosphorylation. Furthermore, this validation strategy could be adaptable to other protein kinases and readily combined with other phosphoproteomic methods.     

Evolved to be active: sulfate ions define substrate recognition sites of CK2alpha and emphasise its exceptional role within the CMGC family of eukaryotic protein kinases

CK2alpha is the catalytic subunit of protein kinase CK2 and a member of the CMGC family of eukaryotic protein kinases like the cyclin-dependent kinases, the MAP kinases and glycogen-synthase kinase 3. We present here a 1.6 A resolution crystal structure of a fully active C-terminal deletion mutant of human CK2alpha liganded by two sulfate ions, and we compare this structure systematically with representative structures of related CMGC kinases. The two sulfate anions occupy binding pockets at the activation segment and provide the structural basis of the acidic consensus sequence S/T-D/E-X-D/E that governs substrate recognition by CK2. The anion binding sites are conserved among those CMGC kinases. In most cases they are neutralized by phosphorylation of a neighbouring threonine or tyrosine side-chain, which triggers conformational changes for regulatory purposes. CK2alpha, however, lacks both phosphorylation sites at the activation segment and structural plasticity. Here the anion binding sites are functionally changed from regulation to substrate recognition. These findings underline the exceptional role of CK2alpha as a constitutively active enzyme within a family of strictly controlled protein kinases.     

Phosphorylation and regulation of a G protein-coupled receptor by protein kinase CK2

We demonstrate a role for protein kinase casein kinase 2 (CK2) in the phosphorylation and regulation of the M3-muscarinic receptor in transfected cells and cerebellar granule neurons. On agonist occupation, specific subsets of receptor phosphoacceptor sites (which include the SASSDEED motif in the third intracellular loop) are phosphorylated by CK2. Receptor phosphorylation mediated by CK2 specifically regulates receptor coupling to the Jun-kinase pathway. Importantly, other phosphorylation-dependent receptor processes are regulated by kinases distinct from CK2. We conclude that G protein-coupled receptors (GPCRs) can be phosphorylated in an agonist-dependent fashion by protein kinases from a diverse range of kinase families, not just the GPCR kinases, and that receptor phosphorylation by a defined kinase determines a specific signalling outcome. Furthermore, we demonstrate that the M3-muscarinic receptor can be differentially phosphorylated in different cell types, indicating that phosphorylation is a flexible regulatory process where the sites that are phosphorylated, and hence the signalling outcome, are dependent on the cell type in which the receptor is expressed.     

Analysis of the CK2-dependent phosphorylation of serine 13 in Cdc37 using a phospho-specific antibody and phospho-affinity gel electrophoresis

The CK2-dependent phosphorylation of Ser13 in cell division cycle protein 37 (Cdc37), a kinase-specific heat shock protein 90 (Hsp90) cochaperone, has previously been reported to be essential for the association of Cdc37 with signaling protein kinases [Bandhakavi S, McCann RO, Hanna DE & Glover CVC (2003) J Biol Chem278, 2829-2836; Shao J, Prince T, Hartson SD & Matts RL (2003) J Biol Chem278, 38117-38220; Miyata Y & Nishida E (2004) Mol Cell Biol24, 4065-4074]. Here we describe a new phospho-specific antibody against Cdc37 that recognizes recombinant purified Cdc37 only when incubated with CK2 in the presence of Mg(2+) and ATP. The replacement of Ser13 in Cdc37 by nonphosphorylatable amino acids abolished binding to this antibody. The antibody was specific for phosphorylated Cdc37 and did not crossreact with other CK2 substrates such as Hsp90 and FK506-binding protein 52. Using this antibody, we showed that complexes of Hsp90 with its client signaling kinases, Cdk4, MOK, v-Src, and Raf1, contained the CK2-phosphorylated form of Cdc37 in vivo. Immunofluorescent staining showed that Hsp90 and the phosphorylated form of Cdc37 accumulated in epidermal growth factor-induced membrane ruffles. We further characterized the phosphorylation of Cdc37 using phospho-affinity gel electrophoresis. Our analyses demonstrated that the CK2-dependent phosphorylation of Cdc37 on Ser13 caused a specific gel mobility shift, and that Cdc37 in the complexes between Hsp90 and its client signaling protein kinases was in the phosphorylated form. Our results show the physiological importance of CK2-dependent Cdc37 phosphorylation and the usefulness of phospho-affinity gel electrophoresis in protein phosphorylation analysis.     

Phosphorylation of the beta-amyloid precursor protein at the cell surface by ectocasein kinases 1 and 2

The beta-amyloid precursor protein (betaAPP) is one of the rare proteins known to be phosphorylated within its ectodomain. We have shown previously that betaAPP can be phosphorylated within secretory vesicles and at the cell surface (Walter, J., Capell, A., Hung, A. Y. , Langen, H., Schn?lzer, M., Thinakaran, G., Sisodia, S. S., Selkoe, D. J., and Haass, C. (1997) J. Biol. Chem. 272, 1896-1903). We have now specifically characterized the phosphorylation of cell surface-located betaAPP and identified two ectoprotein kinases that phosphorylate betaAPP at the outer face of the plasma membrane. By using selective protein kinase inhibitors and by investigating the usage of ATP and GTP as cosubstrates, we demonstrate that membrane-bound betaAPP as well as secreted forms of betaAPP can be phosphorylated by casein kinase (CK) 1- and CK2-like ectoprotein kinases. The ectodomain of betaAPP was also phosphorylated by purified CK1 and CK2 in vitro, but not by protein kinases A and C. Phosphorylation of betaAPP by ectoprotein kinases and by purified CK1 and CK2 occurred within an acidic domain in the N-terminal half of the protein. Heparin strongly inhibited the phosphorylation of cell-surface betaAPP by ecto-CK1 and ecto-CK2, indicating a regulatory role of this extracellular matrix component in betaAPP phosphorylation.     

Ability of CK2β to selectively regulate cellular protein kinases

The Wee1 protein kinase plays a prominent role in keeping cyclin dependent kinase 1 (CDK1) inactive during the G2 phase of the cell cycle. At the onset of mitosis, Wee1 is ubiquitinated by the E3 ubiquitin ligase SCF(beta-TrCP) and subsequently degraded by the proteasome machinery. Previously, it has been reported that although Wee1 lacks the conserved binding motif recognised by beta-TrCP, the CDK-catalysed phosphorylation of Wee1 at Ser123 creates a phosphodegron and primes phosphorylation of two other protein kinases, polo-like kinase 1 (PLK1) and protein kinase CK2, which create two additional phosphodegrons recognised by beta-TrCP. These events contribute to destabilise Wee1 at the onset of mitosis (Watanabe et al. Proc Natl Acad Sci USA 101:4419-4424, 2004). We show here that in addition to the ability of CK2 to phosphorylate Wee1 as reported earlier, the regulatory beta-subunit of protein kinase CK2 can interact with Wee1 in high molecular mass complexes. Indirect immunofluorescence microscopy revealled subcellular co-localisation of CK2beta and Wee1 in the nucleus. Moreover, in vitro phosphorylation assays showed that CK2beta indirectly up-regulates the activity of CDK1 with respect to histone H1 phosphorylation by inhibiting Wee1 kinase. These findings support the view that CK2beta regulates various intracellular processes by modulating the activity of protein kinases that are distinct from CK2 and that protein kinase CK2 plays an important role in events related to the regulation of cell cycle progression as a tetrameric enzyme but also through the individual subunits.     

Unusual phyletic distribution of peptidases as a tool for identifying potential drug targets

Profound changes in the phosphorylation state of many proteins occur during mitosis. It is well established that many of these mitotic phosphorylations are carried out by archetypal mitotic kinases that are activated only during mitosis, shifting the equilibrium of kinases and phosphatases towards phosphorylation. However, many studies have also detailed the phosphorylation of proteins at mitosis by kinases that are constitutively active throughout the cell cycle. In most cases, it is uncertain how kinases and phosphatases that appear to be constitutively active can induce phosphorylations specifically at mitosis. In this issue of the Biochemical Journal, Escargueil and Larsen provide evidence of an interesting alternative mechanism to attain specific mitotic phosphorylation. A mitosis-specific phosphorylation site in DNA topoisomerase IIalpha, which is recognized by the MPM-2 antibody, is phosphorylated by protein kinase CK2. The authors found that phosphorylation of this site is suppressed during interphase due to competing dephosphorylation by protein phosphatase 2A. Interestingly, protein phosphatase 2A is excluded from the nucleus during early mitosis, allowing CK2 to phosphorylate topoisomerase IIalpha. It is possible that similar mechanisms are used to regulate the phosphorylation of other proteins.     

T. cruzi DNA polymerase beta (Tcpolβ) is phosphorylated in vitro by CK1, CK2 and TcAUK1 leading to the potentiation of its DNA synthesis activity

The unicellular protozoan Trypanosoma cruzi is the causing agent of Chagas disease which affects several millions of people around the world. The components of the cell signaling pathways in this parasite have not been well studied yet, although its genome can encode several components able to transduce the signals, such as protein kinases and phosphatases. In a previous work we have found that DNA polymerase β (Tcpolβ) can be phosphorylated in vivo and this modification activates the synthesis activity of the enzyme. Tcpolβ is kinetoplast-located and is a key enzyme in the DNA base excision repair (BER) system. The polypeptide possesses several consensus phosphorylation sites for several protein kinases, however, a direct phosphorylation of those sites by specific kinases has not been reported yet. Tcpolβ has consensus phosphorylation sites for casein kinase 1 (CK1), casein kinase 2 (CK2) and aurora kinase (AUK). Genes encoding orthologues of those kinases exist in T. cruzi and we were able to identify the genes and to express them to investigate whether or no Tcpolβ could be a substrate for in vitro phosphorylation by those kinases. Both CK1 and TcAUK1 have auto-phosphorylation activities and they are able to phosphorylate Tcpolβ. CK2 cannot perform auto-phosphorylation of its subunits, however, it was able to phosphorylate Tcpolβ. Pharmacological inhibitors used to inhibit the homologous mammalian kinases can also inhibit the activity of T. cruzi kinases, although, at higher concentrations. The phosphorylation events carried out by those kinases can potentiate the DNA polymerase activity of Tcpolβ and it is discussed the role of the phosphorylation on the DNA polymerase and lyase activities of Tcpolβ. Taken altogether, indicates that CK1, CK2 and TcAUK1 can play an in vivo role regulating the function of Tcpolβ.     

Casein kinase 2 associates with and phosphorylates dishevelled.

The dishevelled (dsh) gene of Drosophila melanogaster encodes a phosphoprotein whose phosphorylation state is elevated by Wingless stimulation, suggesting that the phosphorylation of Dsh and the kinase(s) responsible for this phosphorylation are integral parts of the Wg signaling pathway. We found that immunoprecipitated Dsh protein from embryos and from cells in tissue culture is associated with a kinase activity that phosphorylates Dsh in vitro. Purification and peptide sequencing of a 38 kDa protein co-purifying with this kinase activity showed it to be identical to Drosophila Casein Kinase 2 (CK2). Tryptic phosphopeptide mapping indicates that identical peptides are phosphorylated by CK2 in vitro and in vivo, suggesting that CK2 is at least one of the kinases that phosphorylates Dsh. Overexpression of Dfz2, a Wingless receptor, also stimulated phosphorylation of Dsh, Dsh-associated kinase activity, and association of CK2 with Dsh, thus suggesting a role for CK2 in the transduction of the Wg signal.     

Pea DNA topoisomerase I is phosphorylated and stimulated by casein kinase 2 and protein kinase C

DNA topoisomerase I catalyzes the relaxation of superhelical DNA tension and is vital for DNA metabolism; therefore, it is essential for growth and development of plants. Here, we have studied the phosphorylation-dependent regulation of topoisomerase I from pea (Pisum sativum). The purified enzyme did not show autophosphorylation but was phosphorylated in an Mg(2+)-dependent manner by endogenous protein kinases present in pea nuclear extracts. This phosphorylation was abolished with calf intestinal alkaline phosphatase and lambda phosphatase. It was also phosphorylated by exogenous casein kinase 2 (CK2), protein kinase C (PKC; from animal sources), and an endogenous pea protein, which was purified using a novel phorbol myristate acetate affinity chromatography method. All of these phosphorylations were inhibited by heparin (inhibitor of CK2) and calphostin (inhibitor of PKC), suggesting that pea topoisomerase I is a bona fide substrate for these kinases. Spermine and spermidine had no effect on the CK2-mediated phosphorylation, suggesting that it is polyamine independent. Phospho-amino acid analysis showed that only serine residues were phosphorylated, which was further confirmed using antiphosphoserine antibody. The topoisomerase I activity increased after phosphorylation with exogenous CK2 and PKC. This study shows that these kinases may contribute to the physiological regulation of DNA topoisomerase I activity and overall DNA metabolism in plants.     

Regulation of ribosomal protein S6 phosphorylation by casein kinase 1 and protein phosphatase 1

Ribosomal protein S6 (rpS6) is a critical component of the 40 S ribosomal subunit that mediates translation initiation at the 5'-m(7)GpppG cap of mRNA. In response to mitogenic stimuli, rpS6 undergoes ordered C-terminal phosphorylation by p70 S6 kinases and p90 ribosomal S6 kinases on four conserved Ser residues (Ser-235, Ser-236, Ser-240, and Ser-244) whose modification potentiates rpS6 cap binding activity. A fifth site, Ser-247, is also known to be phosphorylated, but its function and regulation are not well characterized. In this study, we employed phospho-specific antibodies to show that Ser-247 is a target of the casein kinase 1 (CK1) family of protein kinases. CK1-dependent phosphorylation of Ser-247 was induced by mitogenic stimuli and required prior phosphorylation of upstream S6 kinase/ribosomal S6 kinase residues. CK1-mediated phosphorylation of Ser-247 also enhanced the phosphorylation of upstream sites, which implies that bidirectional synergy between C-terminal phospho-residues is required to sustain rpS6 phosphorylation. Consistent with this idea, CK1-dependent phosphorylation of rpS6 promotes its association with the mRNA cap-binding complex in vitro. Additionally, we show that protein phosphatase 1 (PP1) antagonizes rpS6 C terminus phosphorylation and cap binding in intact cells. These findings further our understanding of rpS6 phospho-regulation and define a direct link between CK1 and translation initiation.     

Identification of a CK2 phosphorylation site in mdm2.

Mdm2 is a cellular oncoprotein the most obvious function of which is the down-regulation of the growth suppressor protein p53. It represents a highly phosphorylated protein but only little is yet known about the sites phosphorylated in vivo, the kinases that are responsible for the phosphorylation or the functional relevance of the phosphorylation status. Recently, we have shown that mdm2 is a good substrate for protein kinase CK2 at least in vitro. Computer analysis of the primary amino acid sequence of mdm2 revealed 19 putative CK2 phosphorylation sites. By using deletion mutants of mdm2 and a peptide library we identified the serine residue at position 269 which lies within a canonical CK2 consensus sequence (EGQELSDEDDE) as the most important CK2 phosphorylation site. Moreover, by using the mdm2 S269A mutant for in vitro phosphorylation assays this site was shown to be phosphorylated by CK2. Binding studies revealed that phosphorylation of mdm2 at S269 does not have any influence on the binding of p53 to mdm2.     

Identification of protein kinase CK2 as a potent kinase of Epstein-Barr virus latent membrane protein 1

The C-terminus of latent membrane protein 1 (LMP1) can be phosphorylated in vivo. However, the protein kinase responsible for LMP1 phosphorylation has not yet been identified. In this study, GST fusion proteins containing the C-terminus of LMP1 were generated and used as substrates to survey the kinases that phosphorylate LMP1. Among several purified protein kinases tested, only protein kinase CK2 (CK2) could specifically phosphorylate LMP1. Using the in-gel kinase assay in the absence and presence of a selective CK2 inhibitor, 4,5,6,7-tetrabromobenzotriazole, CK2 was determined to be the major kinase to phosphorylate LMP1 in lymphoma and epithelial cell lines. This is the first study to show that CK2 is a potent kinase to phosphorylate LMP1 in vitro.     

Chemical dissection of the APC Repeat 3 multistep phosphorylation by the concerted action of protein kinases CK1 and GSK3

A crucial event in machinery controlled by Wnt signaling is the association of beta-catenin with the adenomatous polyposis coli (APC) protein, which is essential for the degradation of beta-catenin and requires the multiple phosphorylation of APC at six serines (1501, 1503, 1504, 1505, 1507, and 1510) within its repeat three (R3) region. Such a phosphorylation is believed to occur by the concerted action of two protein kinases, CK1 and GSK3, but its mechanistic aspects are a matter of conjecture. Here, by combining the usage of variably phosphorylated peptides reproducing the APC R3 region and Edman degradation assisted localization of residues phosphorylated by individual kinases, we show that the process is initiated by CK1, able to phosphorylate S1510 and S1505, both specified by non-canonical determinants. Phosphorylation of S1505 primes subsequent phosphorylation of S1501 by GSK3. In turn, phospho-S1501 triggers the hierarchical phosphorylation of S1504 and S1507 by CK1. Once phosphorylated, S1507 primes the phosphorylation of both S1510 and S1503 by CK1 and GSK3, respectively, thus completing all six phosphorylation steps. Our data also rule out the intervention of CK2 despite the presence of a potential CK2 phosphoacceptor site, S1510LDE, in the R3 repeat. S1510 is entirely unaffected by CK2, while it is readily phosphorylated even in the unprimed peptide by CK1delta but not by CK1gamma. This discloses a novel motif significantly different from non-canonical sequences phosphorylated by CK1 in other proteins, which appears to be specifically recognized by the delta isoform of CK1.     

Comparison of the phosphorylation of microtubule-associated protein tau by non-proline dependent protein kinases

Microtubule-associated protein tau from Alzheimer brain has been shown to be phosphorylated at several ser/thr-pro and ser/thr-X sites (Hasegawa, M. et al., J. Biol. Chem, 267, 17047–17054, 1992). Several proline-dependent protein kinases (PDPKs) (MAP kinase, cdc2 kinase, glycogen synthase kinase-3, tubulin-activated protein kinase, and 40 kDa neurofilament kinase) are implicated in the phosphorylation of the ser-thr-pro sites. The identity of the kinase(s) that phosphorylate that ser/thr-X sites are unknown. To identify the latter kinase(s) we have compared the phosphorylation of bovine tau by several brain protein kinases. Stoichiometric phosphorylation of tau was achieved by casein kinase-1, calmodulin-dependent protein kinase II, Gr kinase, protein kinase C and cyclic AMP-dependent protein kinase, but not with casein kinase-2 or phosphorylase kinase. Casein kinase-1 and calmodulin-dependent protein kinase II were the best tau kinases, with greater than 4 mol and 3 mol³²P incorporated, respectively, into each mol of tau. With the sequential addition of these two kinases,³²P incorporation approached 6 mol. Peptide mapping revealed that the different kinases largely phosphorylate different sites on tau. After phosphorylation by casein kinase-1, calmodulin-dependent protein kinase II, Gr kinase, cyclic AMP-dependent protein kinase and casein kinase-2, the mobility of tau isoforms as detected by SDS-PAGE was decreased. Protein kinase C phosphorylation did not produce such a mobility shift. Our results suggest that one or more of the kinases studied here may participate in the hyperphosphorylation of tau in Alzheimer disease. Such phosphorylation may serve to modulate the activaties of other tau kinases such as the PDPKs.Abbreviations PHF paired helical filaments - A-kinase cyclic AMP-dependent protein kinase - CaM kinase II calcium/calmodulin-dependent protein kinase II - C-kinase calcium-phospholipid-dependent protein kinase - CK-1 casein kinase-1 - CK-2 casein kinase-2 - Gr kinase calcium/calmodulin-dependent protein kinase from rat cerebellum - GSK-3 glycogen synthase kinase-3 - MAP kinase mitogen-activated protein kinase - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Phosphorylation by casein kinase 2 regulates Nap1 localization and function

In Saccharomyces cerevisiae, the evolutionarily conserved nucleocytoplasmic shuttling protein Nap1 is a cofactor for the import of histones H2A and H2B, a chromatin assembly factor and a mitotic factor involved in regulation of bud formation. To understand the mechanism by which Nap1 function is regulated, Nap1-interacting factors were isolated and identified by mass spectrometry. We identified several kinases among these proteins, including casein kinase 2 (CK2), and a new bud neck-associated protein, Nba1. Consistent with our identification of the Nap1-interacting kinases, we showed that Nap1 is phosphorylated in vivo at 11 sites and that Nap1 is phosphorylated by CK2 at three substrate serines. Phosphorylation of these serines was not necessary for normal bud formation, but mutation of these serines to either alanine or aspartic acid resulted in cell cycle changes, including a prolonged S phase, suggesting that reversible phosphorylation by CK2 is important for cell cycle regulation. Nap1 can shuttle between the nucleus and cytoplasm, and we also showed that CK2 phosphorylation promotes the import of Nap1 into the nucleus. In conclusion, our data show that Nap1 phosphorylation by CK2 appears to regulate Nap1 localization and is required for normal progression through S phase.     

Ser/Thr phosphorylation of hematopoietic specific protein 1 (HS1): implication of protein kinase CK2.

Hematopoietic lineage cell-specific protein 1 (HS1), a tyrosine multiphosphorylated protein implicated in receptor-mediated apoptosis and proliferative responses, is shown here to become Ser/Thr phosphorylated upon incubation of platelets with radiolabeled inorganic phosphate. The in vivo Ser/Thr phosphorylation of HS1 is enhanced by okadaic acid and reduced by specific inhibitors of casein kinase (CK)2. In vitro, HS1 is an excellent substrate for either CK2 alpha subunit alone (Km = 47 nM) or CK2 holoenzyme, tested in the presence of polylysine (Km = 400 nM). Phosphorylation reaches a stoichiometry of about 2 mol phosphate per mol HS1 and occurs mainly at threonyl residue(s), mostly located in the N-terminal region, but also at seryl residue(s) residing in the central core of the molecule (208-402), as judged from experiments with deleted forms of HS1. Ser/Thr phosphorylation of HS1, either induced in vivo by okadaic acid or catalysed in vitro by CK2, potentiates subsequent phosphorylation at tyrosyl residues. These data indicate the possibility that regulation of HS1 may also be under the control of Ser/Thr phosphorylation, and suggest that in quiescent cells CK2 could play a role in inducing constitutive Tyr phosphorylation of HS1 in the absence of stimuli that activate the protein tyrosine kinase pathway.     

Are calcium-dependent protein kinases involved in the regulation of glycolytic/gluconeogenetic enzymes? Studies with Ca2+/calmodulin-dependent protein kinase and protein kinase C

Changes in glycolytic flux have been observed in liver under conditions where effects of cAMP seem unlikely. We have, therefore, studied the phosphorylation of four enzymes involved in the regulation of glycolysis and gluconeogenesis (6-phosphofructo-1-kinase from rat liver and rabbit muscle; pyruvate kinase, 6-phosphofructo-2-kinase and fructose-1,6-bisphosphatase from rat liver) by defined concentrations of two cAMP-independent protein kinases: Ca2+/calmodulin-dependent protein kinase and Ca2+/phospholipid-dependent protein kinase (protein kinase C). The results were compared with those obtained with the catalytic subunit of cAMP-dependent protein kinase. The following results were obtained. 1. Ca2+/calmodulin-dependent protein kinase phosphorylates 6-phosphofructo-1-kinase and L-type pyruvate kinase at a slightly lower rate as compared to cAMP-dependent protein kinase. 2. 6-Phosphofructo-1-kinase is phosphorylated by the two kinases at a single identical position. There is no additive phosphorylation. The final stoichiometry is 2 mol phosphate/mol tetramer. The same holds for L-type pyruvate kinase except that the stoichiometry with either kinase or both kinases together is 4 mol phosphate/mol tetramer. 3. Rabbit muscle 6-phosphofructo-1-kinase is phosphorylated by cAMP-dependent protein kinase but not by Ca2+/calmodulin-dependent protein kinase. 4. Fructose-1,6-bisphosphatase from rat but not from rabbit liver is phosphorylated at the same position but at a markedly lower rate by Ca2+/calmodulin-dependent protein kinase when compared to the phosphorylation by cAMP-dependent protein kinase. 5. 6-Phosphofructo-2-kinase is phosphorylated by Ca2+/calmodulin-dependent protein kinase only at a negligible rate. 6. Protein kinase C does not seem to be involved in the regulation of the enzymes examined: only 6-phosphofructo-2-kinase became phosphorylated to a significant degree. In contrast to the phosphorylation by cAMP-dependent protein kinase, this phosphorylation is not associated with a change of enzyme activity. This agrees with our observation that the sites of phosphorylation by the two kinases are different. The results indicate that Ca2+/calmodulin-dependent protein kinase but not protein kinase C could be involved in the regulation of hepatic glycolytic flux under conditions where changes in the activity of cAMP-dependent protein kinase seem unlikely.