Budding and fission yeast casein kinase I isoforms have dual-specificity protein kinase activity.

We have examined the activity and substrate specificity of the Saccharomyces cerevisiae Hrr25p and the Schizosaccharomyces pombe Hhp1, Hhp2, and Cki1 protein kinase isoforms. These four gene products are isotypes of casein kinase I (CKI), and the sequence of these protein kinases predicts that they are protein serine/threonine kinases. However, each of these four protein kinases, when expressed in Escherichia coli in an active form, was recognized by anti-phosphotyrosine antibodies. Phosphoamino acid analysis of 32P-labeled proteins showed phosphorylation on serine, threonine, and tyrosine residues. The E. coli produced forms of Hhp1, Hhp2, and Cki1 were autophosphorylated on tyrosine, and both Hhp1 and Hhp2 were capable of phosphorylating the tyrosine-protein kinase synthetic peptide substrate polymer poly-E4Y1. Immune complex protein kinases assays from S. pombe cells showed that Hhp1-containing precipitates were associated with a protein-tyrosine kinase activity, and the Hhp1 present in these immunoprecipitates was phosphorylated on tyrosine residues. Although dephosphorylation of Hhp1 and Hhp2 by Ser/Thr phosphatase had little effect on the specific activity, tyrosine dephosphorylation of Hhp1 and Hhp2 caused a 1.8-to 3.1-fold increase in the Km for poly-E4Y1 and casein. These data demonstrate that four different CKI isoforms from two different yeasts are capable of protein-tyrosine kinase activity and encode dual-specificity protein kinases.     

Phosphorylation of varicella-zoster virus glycoprotein gpI by mammalian casein kinase II and casein kinase I.

Varicella-zoster virus (VZV) glycoprotein gpI is the predominant viral glycoprotein within the plasma membranes of infected cells. This viral glycoprotein is phosphorylated on its polypeptide backbone during biosynthesis. In this report, we investigated the protein kinases which participate in the phosphorylation events. Under in vivo conditions, VZV gpI was phosphorylated on its serine and threonine residues by protein kinases present within lysates of either VZV-infected or uninfected cells. Because this activity was diminished by heparin, a known inhibitor of casein kinase II, isolated gpI was incubated with purified casein kinase II and shown to be phosphorylated in an in vitro assay containing [gamma-32P]ATP. The same glycoprotein was phosphorylated when [32P]GTP was substituted for [32P]ATP in the protein kinase assay. We also tested whether VZV gpI was phosphorylated by two other ubiquitous mammalian protein kinases--casein kinase I and cyclic AMP-dependent kinase--and found that only casein kinase I modified gpI. When the predicted 623-amino-acid sequence of gpI was examined, two phosphorylation sites known to be optimal for casein kinase II were observed. Immediately upstream from each of the casein kinase II sites was a potential casein kinase I phosphorylation site. In summary, this study showed that VZV gpI was phosphorylated by each of two mammalian protein kinases (casein kinase I and casein kinase II) and that potential serine-threonine phosphorylation sites for each of these two kinases were present in the viral glycoprotein.     

Subcellular localization of casein kinase I

An anti-yeast CKI antiserum was shown to cross-react with CKI isolated from Krebs II mouse ascites tumour cells. The mammalian CKI showed virtually the same molecular mass (app. 45 kDa) as the yeast enzyme. By immunofluorescence it could be shown that CKI is preferably located in the nucleolus.     

Identification of syntaxin-1A sites of phosphorylation by casein kinase I and casein kinase II.

Casein kinases I (CKI) are serine/threonine protein kinases widely expressed in a range of eukaryotes including yeast, mammals and plants. They have been shown to play a role in diverse physiological events including membrane trafficking. CKI alpha is associated with synaptic vesicles and phosphorylates some synaptic vesicle associated proteins including SV2. In this report, we show that syntaxin-1A is phosphorylated in vitro by CKI on Thr21. Casein kinase II (CKII) has been shown previously to phosphorylate syntaxin-1A in vitro and we have identified Ser14 as the CKII phosphorylation site, which is known to be phosphorylated in vivo. As syntaxin-1A plays a key role in the regulation of neurotransmitter release by forming part of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex, we propose that CKI may play a role in synaptic vesicle exocytosis.     

Cytosine-specific type II DNA methyltransferases. A conserved enzyme core with variable target-recognizing domains

Comparisons of the amino acid sequences of m5C DNA methyltransferases (Mtases) from 11 prokaryotes and one eukaryote reveal a very similar organization. Among all the enzymes one can distinguish highly conserved "core" sequences and "variable" regions. The core sequences apparently mediate steps of the methylation reaction that are common to all the enzymes. The major variable region has been shown in our previous studies on multispecific phage Mtases to contain the target-recognizing domains (TRDs) of these enzymes. Here we have compared the amino acid sequences of various TRDs from phage Mtases. This has revealed the presence of both highly conserved and variable amino acids. We postulate that the conserved residues represent a "consensus" sequence defining a TRD, whereas the specificity of the TRD is determined by the variable residues. We have observed similarity between this consensus sequence and sequences in the variable region of the monospecific Mtases. We predict that the regions thus identified represent part of the TRDs of monospecific Mtases.     

The casein kinase I family in Wnt signaling.

The canonical Wnt-signaling pathway is critical for many aspects of development, and mutations in components of the Wnt pathway are carcinogenic. Recently, sufficiency tests identified casein kinase Iepsilon (CKIepsilon) as a positive component of the canonical Wnt/beta-catenin pathway, and necessity tests showed that CKIepsilon is required in vertebrates to transduce Wnt signals. In addition to CKIepsilon, the CKI family includes several other isoforms (alpha, beta, gamma, and delta) and their role in Wnt sufficiency tests had not yet been clarified. However, in Caenorhabditis elegans studies, loss-of-function of a CKI isoform most similar to alpha produced the mom phenotype, indicative of loss-of-Wnt signaling. In this report, we examine the ability of the various CKI isoforms to activate Wnt signaling and find that all the wild-type CKI isoforms do so. Dishevelled (Dsh), another positive component of the Wnt pathway, becomes phosphorylated in response to Wnt signals. All the CKI isoforms, with the exception of gamma, increase the phosphorylation of Dsh in vivo. In addition, CKI directly phosphorylates Dsh in vitro. Finally, we find that CKI is required in vivo for the Wnt-dependent phosphorylation of Dsh. These studies advance our understanding of the mechanism of Wnt action and suggest that more than one CKI isoform is capable of transducing Wnt signals in vivo.     

Association of CPI-17 with protein kinase C and casein kinase I

The protein kinase C-potentiated inhibitor protein of 17kDa, called CPI-17, specifically inhibits myosin light chain phosphatase (MLCP). Phosphorylation of Thr-38 in vivo highly potentiates the ability of CPI-17 to inhibit MLCP. Thr-38 has been shown to be phosphorylated in vitro by a number of protein kinases including protein kinase C (PKC), Rho-associated coiled-coil kinase (ROCK), and protein kinase N (PKN). In this study we have focused on the association of protein kinases with CPI-17. Using affinity chromatography and Western blot analysis, we found interaction with all PKC isotypes and casein kinase I isoforms, CKIalpha and CKI. By contrast, ROCK and PKN did not associate with CPI-17, suggesting that PKC may be the relevant kinase that phosphorylates Thr-38 in vivo. CPI-17 interacted with the cysteine-rich domain of PKC and was phosphorylated by all PKC isotypes. We previously found that CPI-17 co-purified with casein kinase I in brain suggesting they are part of a complex and we now show that CPI-17 associates with the kinase domain of CKI isoforms.     

The casein kinase I family: roles in morphogenesis.

Wnt signals play important roles in development and oncogenesis and are transduced through at least two pathways: a canonical beta-catenin-dependent and a beta-catenin-independent cascade. Casein kinase I (CKI) is required in both invertebrates and vertebrates to transduce canonical Wnt signals. However, its role in the beta-catenin-independent pathway was unknown. During vertebrate embryogenesis, the beta-catenin-independent cascade is thought to control cell movements and has been postulated to be analogous to the Drosophila planar cell polarity pathway, which signals through the JNK cascade. Here, we report that blocking CKI function inhibits embryonic morphogenesis and activates JNK in cell lines. These studies suggest that CKI might also act in the beta-catenin-independent pathway and indicate a role for CKI during convergence extension in early vertebrate development.     

Similarities and differences in the properties of multiple isoforms of maize endosperm casein kinase I (CK-I)

Two novel type I casein kinases named CK-1B and CK-1C have been purified from maize endosperm (three weeks after anthesis) by a six step procedure involving ammonium sulfate precipitation, DEAE-cellulose, Sephadex G-75, Heparin-sepharose, and ATP-agarose chromatography. The catalytic subunits of both enzymes were identified as a 35-37 kDa polypeptide doublet by in situ phosphorylation after SDS/PAGE in active casein gel. Both enzymes required 5-10 mmol · L⁻¹ Mg²⁺ for maximal activity, could utilize only ATP as phosphate donor, were insensitive to heparin, were not autophosphorylated, had a pH optimum at pH 7 to 8.5, and exclusively phosphorylated acidic proteins (casein, phosvitin). Regarding the enzyme differences, their properties were as follows: a) CK-1B could bind on ATP-agarose affinity column, while CK-1C could not; b) the activity of CK-1C was strongly stimulated at low concentrations (1 mmol/L) of spermine, while that of CK-1B was inhibited; c) CK-1B and CK-1C Km values for ATP were 11 μmol · L⁻¹ and 26 μmol · L⁻¹, respectively; d) Mg²⁺ could substituted by Mn²⁺ in the CK-1B catalytic activity (by about 80 percnt;); e) CK-1B phosphorylated serine, while CK-1C both serine and threonine on casein. The combination of these results with those from Babatsikos and Yupsanis (2000) brings the number of investigated maize endosperm CK-I isoforms to three (CK-1B, CK-1C, and CK-1E). This is the first biochemical approach demonstrating that multiple isoforms of CK-I casein kinases are present in the same plant tissue.     

Identification of inhibitory autophosphorylation sites in casein kinase I epsilon.

Casein kinase I epsilon (CKIepsilon) is a widely expressed protein kinase implicated in the regulation of diverse cellular processes including DNA replication and repair, nuclear trafficking, and circadian rhythm. CKIepsilon and the closely related CKIdelta are regulated in part through autophosphorylation of their carboxyl-terminal extensions, resulting in down-regulation of enzyme activity. Treatment of CKIepsilon with any of several serine/threonine phosphatases causes a marked increase in kinase activity that is self-limited. To identify the sites of inhibitory autophosphorylation, a series of carboxyl-terminal deletion mutants was constructed by site-directed mutagenesis. Truncations that eliminated specific phosphopeptides present in the wild-type kinase were used to guide construction of specific serine/threonine to alanine mutants. Amino acids Ser-323, Thr-325, Thr-334, Thr-337, Ser-368, Ser-405, Thr-407, and Ser-408 in the carboxyl-terminal tail of CKIepsilon were identified as probable in vivo autophosphorylation sites. A recombinant CKIepsilon protein with serine and threonine to alanine mutations eliminating these autophosphorylation sites was 8-fold more active than wild-type CKIepsilon using IkappaBalpha as a substrate. The identified autophosphorylation sites do not conform to CKI substrate motifs identified in peptide substrates.     

Multiple functional domains of human U2 small nuclear RNA: strengthening conserved stem I can block splicing.

We showed previously that a branch site mutation in simian virus 40 early pre-mRNA that prevented small t antigen mRNA splicing could be efficiently suppressed by a compensatory mutation in a coexpressed U2 small nuclear (sn) RNA gene. We have now generated second-site mutations in this suppressor gene to investigate regions of U2 RNA required for function. A number of mutations in a putative stem at the 5' end of the molecule inhibited splicing, indicating that bases in this region are important for activity. However, several lines of evidence suggested that formation of the entire stem is not essential for splicing. Indeed, mutations that strengthen the stem actually inhibited splicing, and evidence that this prevents a required base-pairing interaction with U6 snRNA is presented. These results suggest that the relative stabilities of competing intra- and intermolecular base-pairing interactions play an important role in the splicing reaction. Mutations in a conserved single-stranded region immediately 3' to the branch site recognition sequence all inhibited splicing, indicating that this region is required for U2 function, although its exact role remains unknown. Finally, two mutations in the loop of stem IV at the 3' end of the molecule, which destroy the binding site of U2 sn ribonucleoprotein B", prevented small t splicing; this finding contrasts with previous studies which utilized different assay systems. Analysis of the accumulation and subcellular localization of all of the mutant RNAs showed that they were similar to those of the parental suppressor U2 RNA, indicating that the effects observed indeed reflect defects in splicing.     

Functional mapping of conserved, surface-exposed charges of antibody variable domains

Surface-exposed charges can affect protein structure, stability and solubility as well as the kinetics of both the folding process and interaction with binding partners. We have investigated the influence on kinetic interaction parameters of 14 conserved, surface-exposed charges located away from the paratope in the variable domains of two antibodies of different specificity. We found that conserved, surface-exposed, charged framework residues are asymmetrically distributed on opposite faces of both VH and VL domains. Some of the charges play a critical role in protein folding and stability. While electrostatic forces within or close to the binding interface can be used to optimize the association rate, we confirmed the predicted minor effects of charge modifications remote from the binding site. They had no effect on the dissociation rate parameter. Our study demonstrates the role of residues remote from the interaction site in the recognition function as well as the limited effect of surface charge modifications in antibody fragments on kinetic interaction parameters.     

Multiple regulatory domains on the Byr2 protein kinase.

Byr2 protein kinase, a homolog of mammalian mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEKK) and Saccharomyces cerevisiae STE11, is required for pheromone-induced sexual differentiation in the fission yeast Schizosaccharomyces pombe. Byr2 functions downstream of Ste4, Ras1, and the membrane-associated receptor-coupled heterotrimeric G-protein alpha subunit, Gpa1. Byr2 has a distinctive N-terminal kinase regulatory domain and a characteristic C-terminal kinase catalytic domain. Ste4 and Ras1 interact with the regulatory domain of Byr2 directly. Here, we define the domains of Byr2 that bind Ste4 and Ras1 and show that the Byr2 regulatory domain binds to the catalytic domain in the two-hybrid system. Using Byr2 mutants, we demonstrate that these direct physical interactions are all required for proper signaling. In particular, the physical association between Byr2 regulatory and catalytic domains appears to result in autoinhibition, the loss of which results in kinase activation. Furthermore, we provide evidence that Shk1, the S. pombe homolog of the STE20 protein kinase, can directly antagonize the Byr2 intramolecular interaction, possibly by phosphorylating Byr2.     

Alteration of aminoacyl-tRNA synthetase activities by phosphorylation with casein kinase I

The phosphorylation of a highly purified aminoacyl-tRNA synthetase complex from rabbit reticulocytes by the cyclic nucleotide-independent protein kinase, casein kinase I, has been examined, and the effects of phosphorylation on the synthetase activities were determined. The synthetase complex, purified as described (Kellermann, O., Tonetti, H., Brevet, A., Mirande, M., Pailliez, J.-P., and Waller, J.-P. (1982) J. Biol. Chem. 257, 11041-11048), contains seven aminoacyl-tRNA synthetases and four unidentified proteins and is free of endogenous protein kinase activity. Incubation of the complex with casein kinase I in the presence of ATP results in the phosphorylation of four synthetases, namely, glutamyl-, isoleucyl-, methionyl-, and lysyl-tRNA synthetases. Phosphorylation by casein kinase I alters binding of the aminoacyl-tRNA synthetase complex to tRNA-Sepharose. The phosphorylated synthetase complex elutes from tRNA-Sepharose at 190 mM NaCl, while the nonphosphorylated complex elutes at 275 mM NaCl. Phosphorylation by casein kinase I results in a significant inhibition of aminoacylation by the glutamyl-, isoleucyl-, methionyl-, and lysyl-tRNA synthetases; the activities of the nonphosphorylated synthetases remain unchanged. These data indicate that phosphorylation of aminoacyl-tRNA synthetases in the high molecular weight complex alters the activities of these enzymes. One of the unidentified proteins present in the complex (Mr 37,000) is also highly phosphorylated by casein kinase I. From a comparison of the properties and phosphopeptide pattern of this protein with that of casein kinase I, it appears that the Mr 37,000 protein in the synthetase complex is an inactive form of casein kinase I. This observation provides further evidence for a physiological role for casein kinase I in regulating synthetase activities.     

A sea urchin sperm flagellar adenylate kinase with triplicated catalytic domains

The mitochondrion of sea urchin sperm is located at the base of the sperm head, and the flagellum extends from the mitochondrion for approximately 40 microM. These sperm have two known flagellar, non-mitochondrial, enzymatic systems to rephosphorylate ADP. The first involves the phosphocreatine shuttle, where flagellar creatine kinase (Sp-CK) uses phosphocreatine to rephosphorylate ADP. The second system, studied in this report, is adenylate kinase (Sp-AK), which uses 2 ADP to make ATP + AMP. Cloning of Sp-AK shows that, like Sp-CK, Sp-AK has three catalytic domains. Sp-AK localizes along the entire flagellum, and most of it is tightly bound to the axoneme. Sp-AK activity and flagellar motility were studied using demembranated sperm. The specific Sp-AK inhibitor Ap5A blocks enzyme activity with an IC50 of 0.41 microM. In 1 mm ADP, flagella reactivate motility in 5 min; 1 microM Ap5A completely inhibits this reactivation. No inhibition of motility occurs in Ap5A when 1 mm ATP is added to the reactivation buffer. The pH optimum for Sp-AK is 7.7, an internal pH at which sperm are fully motile. The pH optimum for Sp-CK is 6.7, an internal pH at which sperm are immotile. In isolated, detergent-permeabilized flagella, assayed at pH 7.6, the Km for Sp-AK is 0.32 mm and the Vmax is 2.80 microM ATP formed/min/mg of protein. When assayed at pH 7.6, the Sp-CK Km is 0.25 mm and the Vmax 5.25. At the measured in vivo concentrations of ADP of 114 microM, at pH 7.6, the axonemal Sp-AK could contribute approximately 31%, and Sp-CK 69%, of the total non-mitochondrial ATP synthesis associated with the demembranated axoneme. Thus, Sp-AK could contribute substantially to ATP synthesis utilized for motility. Alternatively, Sp-AK could function in the removal of ADP, which is a potent inhibitor of dynein ATPase.