p13suc1 acts in the fission yeast cell division cycle as a component of the p34cdc2 protein kinase.

cdc2+ encodes a protein kinase that is required during both G1 and G2 phases of the cell division cycle in fission yeast. suc1+ is an essential gene that was originally identified as a plasmid-borne sequence that could rescue certain temperature-sensitive cdc2 mutants. To investigate the role of the suc1+ gene product in the cell cycle p13suc1 has been expressed in Escherichia coli and purified. An immunoaffinity purified anti-p13suc1 polyclonal serum has been prepared and used to identify p13suc1 in fission yeast. The abundance of this protein did not alter either during the cell cycle or during entry into stationary phase. p13suc1 was found in yeast lysates in a complex with the cdc2+ gene product. Approximately 5% of cellular p34cdc2 was associated with p13suc1, and this fraction of p34cdc2 was active as a protein kinase. The stability of the complex was disrupted in yeast strains carrying temperature-sensitive alleles of cdc2 that are suppressible by overexpression of suc1+. The level of association between p13suc1 and p34cdc2 was not affected by cell cycle arrest in adverse nutritional conditions. p13suc1 is not a substrate of the p34cdc2 protein kinase. We propose instead that it acts as a regulatory component of p34cdc2 that facilitates interaction with other proteins.     

Mammalian growth-associated H1 histone kinase: a homolog of cdc2+/CDC28 protein kinases controlling mitotic entry in yeast and frog cells.

Mammalian growth-associated H1 histone kinase, an enzyme whose activity is sharply elevated at mitosis, is similar to cdc2+ protein kinase from Schizosaccharomyces pombe and CDC28 protein kinase from Saccharomyces cerevisiae with respect to immunoreactivity, molecular size, and specificity for phosphorylation sites in H1 histone. Phosphorylation of specific growth-associated sites in H1 histone is catalyzed by yeast cdc2+/CDC28 kinase, as shown by the in vitro thermal lability of this activity in extracts prepared from temperature-sensitive mutants. In addition, highly purified Xenopus maturation-promoting factor catalyzes phosphorylation of the same sites in H1 as do the mammalian and yeast kinases. The data indicate that growth-associated H1 kinase is encoded by a mammalian homolog of cdc2+/CDC28 protein kinase, which controls entry into mitosis in yeast and frog cells. Since H1 histone is known to be an in vivo substrate of the mammalian kinase, this suggests that phosphorylation of H1 histone or an H1 histone counterpart is an important component of the mechanism for entry of cells into mitosis.     

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an affinity purified yeast kinase fusion protein containing a V5-epitope tag for read-out. Purified kinase is obtained through culture of a yeast strain optimized for high copy protein production harboring a plasmid containing a Kinase-V5 fusion construct under a GAL inducible promoter. The yeast is grown in restrictive media with a neutral carbon source for 6 hr followed by induction with 2% galactose. Next, the culture is harvested and kinase is purified using standard affinity chromatographic techniques to obtain a highly purified protein kinase for use in the assay. The purified kinase is diluted with kinase buffer to an appropriate range for the assay and the protein microarrays are blocked prior to hybridization with the protein microarray. After the hybridization, the arrays are probed with monoclonal V5 antibody to identify proteins bound by the kinase-V5 protein. Finally, the arrays are scanned using a standard microarray scanner, and data is extracted for downstream informatics analysis^1,2 to determine a high confidence set of protein interactions for downstream validation in vivo.     

Novel yeast protein kinase (YPK1 gene product) is a 40-kilodalton phosphotyrosyl protein associated with protein-tyrosine kinase activity.

Extracts of bakers' yeast (Saccharomyces cerevisiae) contain protein-tyrosine kinase activity that can be detected with a synthetic Glu-Tyr copolymer as substrate (G. Schieven, J. Thorner, and G.S. Martin, Science 231:390-393, 1986). By using this assay in conjunction with ion-exchange and affinity chromatography, a soluble tyrosine kinase activity was purified over 8,000-fold from yeast extracts. The purified activity did not utilize typical substrates for mammalian protein-tyrosine kinases (enolase, casein, and histones). The level of tyrosine kinase activity at all steps of each preparation correlated with the content of a 40-kDa protein (p40). Upon incubation of the most highly purified fractions with Mn-ATP or Mg-ATP, p40 was the only protein phosphorylated on tyrosine. Immunoblotting of purified p40 or total yeast extracts with antiphosphotyrosine antibodies and phosphoamino acid analysis of 32P-labeled yeast proteins fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the 40-kDa protein is normally phosphorylated at tyrosine in vivo. 32P-labeled p40 immunoprecipitated from extracts of metabolically labeled cells by affinity-purified anti-p40 antibodies contained both phosphoserine and phosphotyrosine. The gene encoding p40 (YPK1) was cloned from a yeast genomic library by using oligonucleotide probes designed on the basis of the sequence of purified peptides. As deduced from the nucleotide sequence of YPK1, p40 is homologous to known protein kinases, with features that resemble known protein-serine kinases more than known protein-tyrosine kinases. Thus, p40 is a protein kinase which is phosphorylated in vivo and in vitro at both tyrosine and serine residues; it may be a novel type of autophosphorylating tyrosine kinase, a bifunctional (serine/tyrosine-specific) protein kinase, or a serine kinase that is a substrate for an associated tyrosine kinase.     

p34cdc2 acts as a lamin kinase in fission yeast

The nuclear lamina is an intermediate filament network that underlies the nuclear membrane in higher eukaryotic cells. During mitosis in higher eukaryotes, nuclear lamins are phosphorylated by a mitosis-specific kinase and this induces disassembly of the lamina structure. Recently, p34cdc2 protein kinase purified from starfish has been shown to induce phosphorylation of lamin proteins and disassembly of the nuclear lamina when incubated with isolated chick nuclei suggesting that p34cdc2 is likely to be the mitotic lamin kinase (Peter, M., J. Nakagawa, M. Dorée, J.C. Labbe, and E.A. Nigg. 1990b. Cell. 45:145-153). To confirm and extend these studies using genetic techniques, we have investigated the role of p34cdc2 in lamin phosphorylation in the fission yeast. As fission yeast lamins have not been identified, we have introduced a cDNA encoding the chicken lamin B2 protein into fission yeast. We report here that the chicken lamin B2 protein expressed in fission yeast is assembled into a structure that associates with the nucleus during interphase and becomes dispersed throughout the cytoplasm when cells enter mitosis. Mitotic reorganization correlates with phosphorylation of the chicken lamin B2 protein by a mitosis-specific yeast lamin kinase with similarities to the mitotic lamin kinase of higher eukaryotes. We show that a lamin kinase activity can be detected in cell-free yeast extracts and in p34cdc2 immunoprecipitates prepared from yeast cells arrested in mitosis. The fission yeast lamin kinase activity is temperature sensitive in extracts and immunoprecipitates prepared from strains bearing temperature-sensitive mutations in the cdc2 gene. These results in conjunction with the previously reported biochemical studies strongly suggest that disassembly of the nuclear lamina at mitosis in higher eukaryotic cells is a consequence of direct phosphorylation of nuclear lamins by p34cdc2.     

Purification of MPF from starfish: identification as the H1 histone kinase p34cdc2 and a possible mechanism for its periodic activation

MPF extracted from starfish oocytes copurifies with an M phase-specific H1 histone kinase encoded by a homolog of the fission yeast cell cycle control gene cdc2+. The most purified preparations contain p34cdc2 as the only major protein. Activation of the p34cdc2 kinase is correlated with appearance of the MPF activity both in vivo and in vitro. The increase in protein kinase activity is associated with p34cdc2 dephosphorylation and the decrease in protein kinase activity on leaving M phase with rephosphorylation. Microinjection of a peptide perfectly conserved in p34cdc2 from yeast to humans induces meiotic maturation, suggesting that an inhibitory component in G2 arrested oocytes interacts with this region of the p34cdc2 kinase. We propose that initiation of M phase is brought about by the dephosphorylation of p34cdc2, leading to increase in its protein kinase activity.     

The cell cycle regulator, human p50weel, is a tyrosine kinase and not a serine/tyrosine kinase.

The human weel protein, a homologue of the yeast weel protein, was expressed in E. coli and purified to homogeneity. The purified weel protein phosphorylated the tyrosine residue of cdc2 kinase in HeLa cell extracts in the presence of human cyclin B1. It also phosphorylated the tyrosine but not the threonine residue in the peptide of the amino-terminal of cdc2 kinase, although both these residues have been shown to be phosphorylated in higher eukaryotes in vivo. Furthermore, serine and tyrosine residues of the yeast weel protein are reportedly autophosphorylated in vitro, however the tyrosine residue of the human weel protein was autophosphorylated whereas the serine and threonine residues were not. These data indicate that human p50weel is tyrosine kinase and that it phosphorylated the tyrosine residue of the amino-terminal of cdc2 kinase in the presence of cyclin B1 and that the threonine residue is phosphorylated by another, unknown kinase.     

野生大豆GsPP2CA和GsPKA对GsSnRK1.1蛋白活性的调控

为了明确GsSnRK1.1蛋白激酶在野生大豆生长发育中的具体调控机制,该研究利用酵母二元杂交技术发现了蛋白激酶GsSnRK1.1的互作蛋白GsPP2CA和GsPKA,利用原核表达系统对GsSnRK1.1、GsPP2CA和GsPKA进行了表达和纯化用于Pull down和体外磷酸化分析,并在酵母中研究了GsPP2CA和GsPKA对GsSnRK1.1蛋白活性的调控功能。结果表明：(1)GsSnRK1.1与GsPP2CA和GsPKA具有物理互作关系,Phos Tag和pPKDsub特异性磷酸化抗体检测发现,GsSnRK1.1的Thr176磷酸化可以被蛋白磷酸酶GsPP2CA去磷酸化,GsPKA可能会磷酸化GsSnRK1.1的其他潜在磷酸化位点,进而竞争性抑制GsSnRK1.1的Thr176位点的磷酸化水平。(2)将这些基因回补进入酵母ARY330( snf1/ reg1/ sit4)突变株系中,发现共转化GsSnRK1.1和GsPP2CA或GsPKA的转化子可在非葡萄糖碳源和高葡萄糖碳源的选择培养基上正常生长,GsPP2CA、GsPKA可以替代Reg1和Sit4降低GsSnRK1.1过度磷酸化对酵母细胞产生的毒害作用,进而调控GsSnRK1.1对非发酵型碳源的利用。     

Identification of a glycogen synthase phosphatase from yeast Saccharomyces cerevisiae as protein phosphatase 2A.

A glycogen synthase phosphatase was purified from the yeast Saccharomyces cerevisiae. The purified yeast phosphatase displayed one major protein band which coincided with phosphatase activity on nondenaturing polyacrylamide gel electrophoresis. This phosphatase had a molecular mass of about 160,000 Da determined by gel filtration and was comprised of three subunits, termed A, B, and C. The subunit molecular weights estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 60,000 (A), 53,000 (B), and 37,000 (C), indicating that this yeast glycogen synthase phosphatase is a heterotrimer. On ethanol treatment, the enzyme was dissociated to an active species with a molecular weight of 37,000 estimated by gel filtration. The yeast phosphatase dephosphorylated yeast glycogen synthase, rabbit muscle glycogen phosphorylase, casein, and the alpha subunit of rabbit muscle phosphorylase kinase, was not sensitive to heat-stable protein phosphatase inhibitor 2, and was inhibited 90% by 1 nM okadaic acid. Dephosphorylation of glycogen synthase, phosphorylase, and phosphorylase kinase by this yeast enzyme could be stimulated by histone H1 and polylysines. Divalent cations (Mg2+ and Ca2+) and chelators (EDTA and EGTA) had no effect on dephosphorylation of glycogen synthase or phosphorylase while Mn2+ stimulated enzyme activity by approximately 50%. The specific activity and kinetics for phosphorylase resembled those of mammalian phosphatase 2A. An antibody against a synthetic peptide corresponding to the carboxyl terminus of the catalytic subunit of rabbit skeletal muscle protein phosphatase 2A reacted with subunit C of purified yeast phosphatase on immunoblots, whereas the analogous peptide antibody against phosphatase 1 did not. These data show that this yeast glycogen synthase phosphatase has structural and catalytic similarity to protein phosphatase 2A found in mammalian tissues.     

Expression in Escherichia coli of BCY1, the regulatory subunit of cyclic AMP-dependent protein kinase from Saccharomyces cerevisiae. Purification and characterization

The regulatory (R) subunit of cAMP-dependent protein kinase from the yeast Saccharomyces cerevisiae was expressed in Escherichia coli by engineering the gene for yeast R, BCY1, into an E. coli expression vector that contained a promoter from phage T7. Oligonucleotide-directed mutagenesis was used to create an NdeI restriction site at the natural ATG of the yeast R. This facilitated construction of the T7 expression vector so that the sequence of the protein produced was identical to the natural R subunit. Yeast R was highly expressed in a soluble form. 20 mg of purified yeast R was obtained from 4 liters of E. coli. N-terminal amino acid sequencing revealed that the expressed protein began with the natural sequence. 60% of the molecules contained an N-terminal methionine, and 40% initiated with valine, the second amino acid of yeast R. The protein produced in E. coli migrated on a sodium dodecyl sulfate-polyacrylamide gel with an Mr of 52,000. The yeast R bound 2 mol of cAMP/mol of R monomer with a Kd of 76 nM. The protein was treated with urea to remove bound cAMP. Sedimentation values before and after the urea treatment were identical (s20,w = 5.1). Addition of purified R subunit to a preparation of yeast C subunit (TPK1) rendered catalytic activity cAMP-dependent with an activity ratio of 4.6. The yeast R was autophosphorylated by yeast C to a level of 0.8 mol of phosphate/mol of R monomer. By these criteria, the R subunit produced in E. coli was structurally and functionally identical to the natural yeast R subunit and similar to mammalian type II R subunits.     

Xenopus MAP kinase activator is a serine/threonine/tyrosine kinase activated by threonine phosphorylation.

Xenopus MAP kinase activator, a 45 kDa protein, has been shown to function as a direct upstream factor sufficient for full activation and both tyrosine and serine/threonine phosphorylation of inactive MAP kinase. We have now shown by using an anti-MAP kinase activator antiserum that MAP kinase activator is ubiquitous in tissues and is regulated post-translationally. Activation of MAP kinase activator is correlated precisely with its threonine phosphorylation during the oocyte maturation process. It is a key question whether MAP kinase activator is a kinase or not. We have shown that Xenopus MAP kinase activator purified from mature oocytes is capable of undergoing autophosphorylation on serine, threonine and tyrosine residues. Dephosphorylation of purified activator by protein phosphatase 2A treatment inactivates its autophosphorylation activity as well as its activator activity. Thus, Xenopus MAP kinase activator is a protein kinase with specificity for both serine/threonine and tyrosine. Partial protein sequencing of purified activator indicates that it contains a sequence homologous to kinase subdomains VI and VII of two yeast protein kinases, STE7 and byrl.     

XA21和PI-D2激酶蛋白质在酿酒酵母中的表达及其自我磷酸化研究

白叶枯病和稻瘟病是最主要的水稻病害。Xa21是水稻白叶枯病抗性基因,Pi-d2是稻瘟病抗性基因,二者都编码类受体激酶蛋白质。在前期研究中,曾系统地研究了细菌中表达XA21激酶蛋白质的生化活性。在此实验中利用真核表达系统酿酒酵母对Xa21和Pi-d2编码的蛋白激酶进行了表达、纯化及自我磷酸化活性分析,为进一步的生化分析、蛋白质-蛋白质相互作用研究、底物筛选等奠定了基础。     

Multiple Ca2+/calmodulin-dependent protein kinase genes in a unicellular eukaryote.

We purified a Ca2+/calmodulin (CaM)-dependent protein kinase (CaM kinase) from the yeast Saccharomyces cerevisiae with properties similar to mammalian type II CaM kinases. Degenerate oligonucleotides designed on the basis of the amino acid sequence of tryptic peptides from the 55 kd subunit of the yeast CaM kinase were used to isolate its gene from a set of lambda gt11-yeast genomic DNA phage clones initially selected by the ability to bind 125I-labelled yeast CaM. The cloned gene (CMK1) encodes an open reading frame that is homologous to the sequences of vertebrate type II CaM kinases. Several criteria demonstrated that the CMK1 gene product is the 55 kd polypeptide. Neither over-production (11-fold) nor complete elimination of the CMK1 gene product had any detectably deleterious effect on yeast cell growth. Extracts from cmk1 delta cells, which lacked detectable p55 using an antiserum raised against a Staphylococcus aureus protein A-CMK1 fusion protein, possessed significant residual Ca2+/CAM-dependent protein kinase activity. Using the CMK1 gene as a probe at low stringency, a second gene (CMK2) encoding another CaM-dependent protein kinase with striking sequence similarity to CMK1 was cloned. Deletion of CMK2, or both CMK1 and CMK2, was not lethal, although loss of CMK2 caused a slow rate of spore germination.     

Phosphorylation of yeast DNA-dependent RNA polymerases in vivo and in vitro. Isolation of enzymes and identification of phosphorylated subunits.

Yeast DNA-dependent RNA polymerases I, II, and III are phosphorylated in vivo. Yeast cells were grown continuously in 32Pi and the RNA polymerases were isolated by a new procedure which allows the simultaneous purification of these enzymes from small quantities (35 to 60 g) of cells. Each of the RNA polymerases was phosphorylated. The following phosphorylated polymerase polypeptides were identified: polymerase I subunits of 185,000, 44,000, 36,000, 24,000, and 20,000 daltons; a polymerase II subunit of 24,000 daltons; and polymerase III subunits of 24,000 and 20,000 daltons. The incorporated 32P was acid-stable but base-labile. Phosphoserine and phosphothreonine were identified after partial acid hydrolysis of purified [32P]polymerase I. A yeast protein kinase that co-purifies with polymerase I during part of the isolation procedure was partially purified and characterized. This protein kinase phosphorylates the subunits of the purified polymerases that are phosphorylated in vivo and, in addition, a polymerase I subunit of 48,000 daltons and a polymerase II subunit of 33,500 daltons. Phosphorylation of the purified enzymes with this protein kinase had no substantial effect on polymerase activity in simple assays using native yeast DNA as a template. Preincubation of purified polymerase I with acid or alkaline phosphatase also had no detectable effect on polymerase activity.     

Characterization of the yeast Cdc7p/Dbf4p complex purified from insect cells. Its protein kinase activity is regulated by Rad53p

The yeast Saccharomyces cerevisiae Cdc7p/Dbf4p protein kinase complex was purified to near homogeneity from insect cells. The complex efficiently phosphorylated yeast Mcm2p and less efficiently the remaining Mcm proteins or other replication proteins. Significantly, when pretreated with alkaline phosphatase, Mcm2p became completely inactive as a substrate, suggesting that it must be phosphorylated by other protein kinase(s) to be a substrate for the Cdc7p/Dbf4p complex. Mutant Cdc7p/Dbf4p complexes containing either Cdc7-1p or Dbf4-1 approximately 5p were also partially purified from insect cells and characterized in vitro. Furthermore, the autonomously replicating sequence binding activity of various dbf4 mutants was also analyzed. These studies suggest that the autonomously replicating sequence-binding and Cdc7p protein kinase activation domains of Dbf4p collaborate to form an active Cdc7p/Dbf4p complex and function during S phase in S. cerevisiae. It is shown that Rad53p phosphorylates the Cdc7p/Dbf4p complex in vitro and that this phosphorylation greatly inhibits the kinase activity of Cdc7p/Dbf4p. This result suggests that Rad53p controls the initiation of chromosomal DNA replication by regulating the protein kinase activity associated with the Cdc7p/Dbf4p complex.     

Detection of a mammalian histone H4 kinase that has yeast histidine kinase-like enzymic activity

A well characterized histidine kinase purified from yeast has been shown to phosphorylate histone H4 on a histidine residue. This enzyme is unlike the two-component histidine kinases predominantly found in prokaryotes. Until now, a histidine kinase similar to this yeast enzyme has not been purified from a mammalian source. By using a purification scheme similar to that used to purify the yeast histidine kinase, a protein fraction with histone H4 kinase activity has been isolated from porcine thymus. The yeast histidine kinase was shown to be detectable using an in-gel kinase assay system and using this system, four major bands of histone H4 kinase activity were apparent in the porcine thymus preparation. Through the use of immunoprecipitation, alkaline hydrolysis and subsequent phosphoamino acid analysis it has been demonstrated that this partially purified kinase fraction is capable of phosphorylating histone H4 on histidine. In conclusion, an preparation has been made from porcine thymus that contains histone H4 kinase activity and at least one of the kinases present in this preparation is a histidine kinase.     

Isolation and expression of a pea vicilin cDNA in the yeast Saccharomyces cerevisiae.

A cDNA clone containing the complete coding sequence for vicilin from pea (Pisum sativum L.) was isolated. It specifies a 50,000-Mr protein that in pea is neither post-translationally processed nor glycosylated. The cDNA clone was expressed in yeast from a 2 micron plasmid by using the yeast phosphoglycerate kinase promoter and initiator codon. The resultant fusion protein, which contains the first 16 amino acid residues of phosphoglycerate kinase in addition to the vicilin sequence, was purified and subsequently characterized. It has slightly slower mobility on SDS/polyacrylamide-gel electrophoresis than standard pea vicilin and forms a mixture of multimers, some of which resemble the native protein.     

Two yeast genes encoding calmodulin-dependent protein kinases. Isolation, sequencing and bacterial expressions of CMK1 and CMK2

We have isolated two genes from Saccharomyces cerevisiae that both encode a calmodulin-dependent protein kinase (CaM kinase). The CMK1 gene has been cloned by hybridization using an oligonucleotide probe synthesized on the basis of the peptide sequence of purified yeast CaM kinase (Londesborough, J. (1989) J. Gen. Microbiol. 135, 3373-3383). The other gene, CMK2, which is homologous to CMK1, has been isolated by screening at low stringency with a CMK1 fragment as a probe. The CMK2 product expressed in bacteria shows Ca(2+)- and CaM-dependent protein kinase activity, indicating that CMK2 also encodes a CaM kinase. The CMK1 and CMK2 products expressed in bacteria were found to have different biochemical properties in terms of autoregulatory activity and preference for yeast CaM or bovine CaM for maximal activity. Antibody raised against a peptide fragment of the CMK1 protein cross-reacts with the CMK2 product. Immunoblotting with this antibody indicated that the CMK1 and CMK2 products have apparent molecular masses of 56 and 50 kDa, respectively, in yeast cells. The predicted amino acid sequences of the two CMK products exhibit highest similarity with mammalian calmodulin-dependent multifunctional protein kinase II (CaM kinase II): the similarity within the N-terminal catalytic domain is about 40%, whereas that within the rest of the sequence is 25%. These data indicate that yeast has two kinds of genes encoding CaM kinase isozymes whose structural and functional properties are closely related to those of mammalian CaM kinase II. Another gene may be substituted for function of the CMK1 and CMK2 kinase in vivo, since elimination of both kinase genes is not lethal.     

PKA from Saccharomyces cerevisiae can be activated by cyclic AMP and cyclic GMP.

Analysis of Saccharomyces cerevisiae genome revealed no sequence homologous to cyclic GMP (cGMP) dependent protein kinase from other organisms. Here we demonstrate that cyclic AMP (cAMP) dependent protein kinase purified from S. cerevisiae was almost equally activated by cAMP and cGMP in 3 x 10(-6) M concentrations of either nucleotide in the presence of Mg2+ ions. Interestingly, if Mn2+ ions were used instead of Mg2+, cGMP was only 30% as effective as cAMP in the activation of cAMP-dependent protein kinase. Analogs of cAMP such as 8-chloro-cAMP and 3':5'-cyclic monophosphate of ribofuranosylbenzimidazole were as potent as cAMP in the enzyme activation, while N6,2'-O-dibutyryl-cAMP activated the enzyme to a lower extent. It was also found that yeast cAMP-dependent protein kinase can be activated by limited proteolytic digestion. The results presented were obtained with protamine and ribosomal protein S10 used as phosphorylation substrates.     

A minor species of a type I casein kinase from yeast phosphorylating threonine residues of protein substrate

Protein kinase of Mr 23 000 was isolated from yeast and purified to apparent homogeneity. The enzyme preferentially phosphorylated casein and phosvitin in the presence of ATP as a phosphoryl donor. Its activity was neither affected by cyclic nucleotides nor by heparin. The kinase displayed practically the same substrate specificity as a typical casein kinase I from yeast (Kudlicki, W., Szyszka, R., Paleń, E. and Gasior, E. (1980) Biochim. Biophys. Acta 633, 376-385) except that it phosphorylated threonine instead of serine residues in protein substrates.