(1/481) Optimal sequences for non-phosphate-directed phosphorylation by protein kinase CK1 (casein kinase-1)--a re-evaluation.
A variety of synthetic peptides derived from either the inhibitor-2 (I-2) phosphoacceptor sites or the optimal sequences selected in an oriented peptide library have been compared for their susceptibility to phosphorylation by protein kinase CK1 (also termed casein kinase-1). The I-2-derived peptides are by far preferred over the library peptides by both rat liver CK1 (and by the alpha/beta, gamma and delta/epsilon isoforms immunoprecipitated from it) and recombinant Xenopus laevis CK1 alpha. The superiority of the I-2-derived peptides over the library ones is reflected by Vmax values one to two orders of magnitude higher while the Km values are comparable. Individual substitutions of any of the aspartic acids with alanine in the I-2-derived peptide RRKHAAIGDDDDAYSITA is detrimental, producing both a fall in Vmax and an increase in Km which are more pronounced at position n -3, but also quite significant at positions n -4, n -5 and, to a lesser extent, n -6. The unfavourable effect of these substitutions is more evident with rat liver CK1 than with recombinant Xenopus laevis CK1 alpha. The chimeric peptide IGDDDDAY-S-IIIFFA, resulting from the combination of the N-terminal acidic sequence of the I-2 (Ser86) site and the C-terminal hydrophobic cluster selected in the library peptides (MAEFDTG-S-IIIFFAKKK and MAYYDAA-S-IIIFFAKKK) is phosphorylated as efficiently as the I-2-derived peptide in terms of both Km and Vmax. These combined data strongly support the conclusion that, at variance with the optimal sequences selected in the library, optimal non-phosphate-directed phosphorylation of peptide substrates by CK1 critically relies on the presence of a cluster of acidic residues (preferably aspartic acid) upstream from position n -2, while the highly hydrophobic region downstream from serine selected in the library appears to be dispensable. The reason for these discrepancies remains unclear. The possibility that the library data are biased by the invariant elements forming its scaffold (MA-x-x-x-x-x-SI-x-x-x-x-AKKK) would be consistent with the observation that the library-selected peptides, despite their low Km values, fail to compete against the phosphorylation of protein and peptide substrates by CK1, suggesting that they bind to elements partially distinct from those responsible for substrate recognition. (+info)
(2/481) The Yck2 yeast casein kinase 1 isoform shows cell cycle-specific localization to sites of polarized growth and is required for proper septin organization.
Casein kinase 1 protein kinases are ubiquitous and abundant Ser/Thr-specific protein kinases with activity on acidic substrates. In yeast, the products of the redundant YCK1 and YCK2 genes are together essential for cell viability. Mutants deficient for these proteins display defects in cellular morphogenesis, cytokinesis, and endocytosis. Yck1p and Yck2p are peripheral plasma membrane proteins, and we report here that the localization of Yck2p within the membrane is dynamic through the cell cycle. Using a functional green fluorescent protein (GFP) fusion, we have observed that Yck2p is concentrated at sites of polarized growth during bud morphogenesis. At cytokinesis, GFP-Yck2p becomes associated with a ring at the bud neck and then appears as a patch of fluorescence, apparently coincident with the dividing membranes. The bud neck association of Yck2p at cytokinesis does not require an intact septin ring, and septin assembly is altered in a Yck-deficient mutant. The sites of GFP-Yck2p concentration and the defects observed for Yck-deficient cells together suggest that Yck plays distinct roles in morphogenesis and cytokinesis that are effected by differential localization. (+info)
(3/481) A casein kinase I motif present in the cytoplasmic domain of members of the tumour necrosis factor ligand family is implicated in 'reverse signalling'.
We have identified a putative signalling feature of the cytoplasmic domains of the tumour necrosis factor (TNF) family members based on available amino acid sequence data. A casein kinase I (CKI) consensus sequence is conserved in the cytoplasmic domain of six of 15 members of the type II integral membrane TNF ligand family. We examined the phosphorylation state of transmembrane tumour necrosis factor-alpha (mTNF) with [32P]orthophosphate labelling and in vitro kinase assays, in lipopolysaccharide-stimulated RAW264.7 cells. A dimeric form of the type I soluble TNF receptor (sTNFR) was found to dephosphorylate mTNF. This effect could be prevented by treatment with phosphatase inhibitors. Recombinant CKI was able to phosphorylate mTNF that had been dephosphorylated by sTNFR ligation in vivo, and this was less effective if phosphatase inhibitors had been used to prevent mTNF dephosphorylation. A mutated form of mTNF, lacking the CKI recognition site, cannot be phosphorylated by the enzyme. Binding of sTNFR to mTNF induced an increase in intracellular calcium levels in RAW264.7 cells, implying the presence of an associated signalling pathway. We predict that this CKI motif is phosphorylated in other TNF ligand members, and that it represents a new insight into the mechanism of 'reverse signalling' in this cytokine family. (+info)
(4/481) Insertion of a casein kinase recognition sequence induces phosphorylation of ovine beta-lactoglobulin in transgenic mice.
We have shown that the cellular mechanisms of the mammary gland can be used to produce a phosphorylated form of a normally unphosphorylated milk protein. This was achieved by the insertion of a beta-casein DNA sequence coding for a group of mammary gland casein kinase recognition sites into ovine beta-lactoglobulin. Transgenic mice carrying this modified gene were generated and lactating females were shown to produce a novel beta-lactoglobulin in their milk. The infrared spectrum, reactivity to antiphosphoserine antibody and reduction of electrophoretic mobility on treatment with alkaline phosphatase showed that the novel protein recovered from the milk whey (serum) was phosphorylated and molecular mass determination by mass spectrometry was consistent with the phosphorylation of one or two residues. A similar level of phosphorylation was measured by quantitative infrared spectroscopy. Centrifugation of the milk to pellet the casein micelles showed that most of the phosphorylated beta-lactoglobulin was in the whey and hence not incorporated into casein micelles. (+info)
(5/481) Alterations in C3 activation and binding caused by phosphorylation by a casein kinase released from activated human platelets.
A casein kinase released from activated human platelets phosphorylates a number of plasma proteins extracellularly, and that activation of platelets in systemic lupus erythematosus patients parallels an increase in the phosphate content of plasma proteins, including C3. The present study was undertaken to characterize this platelet protein kinase and to further elucidate the effect(s) on C3 function of phosphorylation by platelet casein kinase. The phosphate content of human plasma C3 was increased from 0.15 to 0.60 mol phosphate/mol of C3 after platelet activation in whole blood or platelet-rich plasma. The platelet casein kinase was distinct from other casein kinases in terms of its dependence on cations, inhibition by specific protein kinase inhibitors, and immunological reactivity. C3 that had been phosphorylated with platelet casein kinase was tested for its susceptibility to cleavage by trypsin or the classical and alternative pathway convertases and its binding to EAC and IgG. Phosphorylation did not affect the cleavage of C3 into C3a and C3b, but the binding of fragments from phosphorylated C3 to EAC14oxy2 cells and to IgG in purified systems and in serum was increased by 1.6-4.5 times over that of unphosphorylated C3. A covariation was seen between the enhanced binding of C3 fragments to IgG after phosphorylation and an increased ratio of glycerol/glycine binding, from 2.0 for unphosphorylated C3 to 4.9 for phosphorylated C3. The present study suggests that an overall effect of phosphorylation of C3 by platelet casein kinase is to enhance the opsonization of immune complexes. (+info)
(6/481) Angiotensin II stimulates serine phosphorylation of the adaptor protein Nck: physical association with the serine/threonine kinases Pak1 and casein kinase I.
Nck is a small adaptor protein consisting exclusively of three SH3 domains and one SH2 domain. Nck is thought to have an important role in cell signalling by coupling receptor tyrosine kinases, via its SH2 domain, to downstream SH3-binding effectors. We report here that angiotensin II, working through the AT1 receptor subtype, stimulates the phosphorylation of Nck in rat aortic smooth muscle cells. Phosphopeptide mapping analysis revealed that Nck is phosphorylated on four peptides containing exclusively phosphoserine in quiescent cells. Treatment with angiotensin II resulted in increased phosphorylation of these four peptides, without the appearance of new phosphopeptides. We show that Nck, via its SH3 domains, specifically binds three major phosphoproteins of 95, 82 and 66 kDa both in vitro and in intact cells. Notably, the phosphorylation of these Nck-binding proteins was found to increase in parallel with that of Nck on stimulation by angiotensin II. One candidate for the 66 kDa phosphoprotein is the serine/threonine kinase p21-activated kinase 1 (Pak1), which was found to form a stable complex with Nck in aortic smooth muscle cells. We have also identified the gamma2 isoform of casein kinase I as another protein kinase that associates with Nck in these cells. These findings indicate that Nck is a target of G-protein-coupled receptors and suggest a role for Pak1 and casein kinase I-gamma2 in downstream signalling or regulation of the AT1 receptor. (+info)
(7/481) Serine phosphorylation of the ligand-activated beta-platelet-derived growth factor receptor by casein kinase I-gamma2 inhibits the receptor's autophosphorylating activity.
Platelet-derived growth factor (PDGF) receptors (PDGFRs) are membrane protein-tyrosine kinases that, upon activation, become tyrosine-phosphorylated and associate with numerous SH2 domain-containing molecules involved in mediating signal transduction. In Rat-2 fibroblasts, we have characterized the phosphorylation of the beta-PDGFR following its activation by PDGF. In contrast to tyrosine phosphorylation, which was transient and returned to near basal levels by 30 min, PDGF-stimulated Ser/Thr phosphorylation of the beta-PDGFR was increased by 5 min and remained elevated after 30 min. In vivo, after 5 min of PDGF stimulation, serine phosphorylation of the beta-PDGFR was greatly reduced by CKI-7, a specific inhibitor of casein kinase I (CKI). In vitro, recombinant CKI-gamma2 phosphorylated the ligand-activated beta-PDGFR on serine residues in a CKI-7-sensitive manner and resulted in a marked inhibition of the receptor's autophosphorylating activity. Furthermore, in Rat-2 fibroblasts, expression of hemagglutinin epitope-tagged active CKI-gamma2 resulted in a dramatic decrease in the tyrosine phosphorylation state of the beta-PDGFR in response to PDGF, consistent with receptor inactivation. Our data suggest that upon PDGF stimulation, CKI-gamma2 is activated and/or translocated in proximity to the beta-PDGFR, whereby it phosphorylates the beta-PDGFR on serine residues and negatively regulates its tyrosine kinase activity, leading to receptor inactivation. (+info)
(8/481) The casein kinase Ialpha isoform is both physically positioned and functionally competent to regulate multiple events of mRNA metabolism.
Casein kinase I is a highly conserved family of serine/threonine protein kinases present in every organism tested from yeast to humans. To date, little is known about the function of the higher eukaryotic isoforms in this family. The CKI isoforms in Saccharomyces cerevisiae, however, have been genetically linked to the regulation of DNA repair, cell cycle progression and cytokinesis. It has also been established that the nuclear localization of two of these isoforms is essential for their function. The work presented here demonstrates that the higher eukaryotic CKIalpha isoform is also present within nuclei of certain established cell lines and associated with discrete nuclear structures. The nature of its nuclear localization was characterized. In this regard, CKIalpha was shown to colocalize with factors involved in pre-mRNA splicing at nuclear speckles and that its association with these structures exhibited several biochemical properties in common with known splicing factors. The kinase was also shown to be associated with a complex that contained certain splicing factors. Finally, in vitro, CKIalpha was shown to be capable of phosphorylating particular splicing factors within a region rich in serine/arginine dipeptide repeat motifs suggesting that it has both the opportunity and the capacity to regulate one or more steps of mRNA metabolism. (+info)