Role of threonine residues in the regulation of manganese-dependent arabidopsis serine/threonine/tyrosine protein kinase activity

Tyrosine phosphorylation in plants could be performed only by dual-specificity kinases. Arabidopsis thaliana dual-specificity protein kinase (AtSTYPK) exhibited strong preference for manganese over magnesium for its kinase activity. The kinase autophosphorylated on serine, threonine and tyrosine residues and phosphorylated myelin basic protein on threonine and tyrosine residues. The AtSTYPK harbors manganese dependent serine/threonine kinase domain, COG3642. His248 and Ser265 on COG3642 are conserved in AtSTYPK and the site-directed mutant, H248A showed loss of serine/threonine kinase activity. The protein kinase activity was abolished when Thr208 in the TEY motif and Thr293 of the activation loop were converted to alanine. The conversion of Thr284 in the activation loop to alanine resulted in an increased phosphorylation. This study reports the first identification of a manganese dependent dual-specificity kinase and the importance of Thr208, Thr284, and Thr293 residues in the regulation of kinase activity.     

Identification and characterization of a myristylated and palmitylated serine/threonine protein kinase.

We report the molecular cloning and initial characterization of a novel fatty acid acylated serine/threonine protein kinase. The putative open reading frame is predicted to encode a 305 amino acid protein possessing a carboxy-terminal protein kinase domain and amino-terminal myristylation and palmitylation sites. The protein kinase has been accordingly denoted as the myristylated and palmitylated serine/threonine protein kinase (MPSK). Human and mouse MPSKs share approximately 93% identity at the amino acid level with complete retention of acylation sites. Radiation hybridization localized the human MPSK gene to chromosome 2q34-37. Northern analysis demonstrated that the human MPSK 1.7 kilobase mRNA is widely distributed. Epitope tagged human MPSK was found to be acylated by myristic acid at glycine residue 2 and by palmitic acid at cysteines 6 and/or 8. Palmitylation of MPSK in these experiments was found to require an intact myristylation site. While epitope tagged MPSK in immune complexes or purified human glutathione S transferase-MPSK was found to autophosphorylate at one or more threonine residues, the enzyme was not found to phosphorylate several other common exogenous substrates. Indeed, only PHAS-I was identified as an exogenous substrate which was found to be phosphorylated on threonine and serine residues.     

Molecular cloning, expression and characterization of the human serine/threonine kinase Akt-3.

Akt (also known as PKB or RAC-PK) is an intracellular serine/threonine kinase involved in regulating cell survival. Although this makes it a promising target for the discovery of drugs to treat human cancer, a complicating factor may be the role played by Akt in insulin signalling. Two human isoforms, Akt-1 and Akt-2, have been described previously and a third isoform has been identified in rats (here termed Akt-3, but also called RAC-PK-gamma or PKB-gamma). We describe the identification of the corresponding human isoform of Akt-3. The gene encoding human Akt-3 was localized to chromosome 1q43-44. The predicted protein sequence is 83% identical to human Akt-1 and 78% identical to human Akt-2, and contains a pleckstrin homology domain and a kinase domain. In contrast to the published rat Akt-3 isoform, human and mouse Akt-3 also possess a C-terminal 'tail' that contains a phosphorylation site (Ser472) thought to be involved in the activation of Akt kinases. In addition to phosphorylation of Ser472, phosphorylation of Thr305 also appears to contribute to the activation of Akt-3 because mutation of both these residues to aspartate increased the catalytic activity of Akt-3, whereas mutation to alanine inhibited activation. Akt-3 activity could be inhibited by the broad spectrum kinase inhibitor staurosporine and by the PKC inhibitor Ro 31-8220, but not by other PKC or PKA inhibitors tested. Although Akt-3 is expressed widely, it is not highly expressed in liver or skeletal muscle, suggesting that its principle function may not be in regulating insulin signalling. These observations suggest that Akt-3 is a promising target for the discovery of novel chemotherapeutic agents which do not interfere with insulin signalling.     

Isolation and characterization of a serine/threonine protein kinase SOS2 gene from Brassica napus

A full-length cDNA of a new serine/threonine (Ser/Thr) protein kinase gene, designated as BnSOS2 (GenBank Acc. No.AY310413), was cloned from Brassica napus by rapid amplification of cDNA ends (RACE). The full-length cDNA of BnSOS2 was 1779 bp and contained a 1539-bp open reading frame encoding a protein of 512 amino acids. Homology analysis shows that BnSOS2 strongly resembles other Ser/Thr protein kinase genes, and that its putative protein belongs to a typical Ser/Thr kinase family. Northern blot analysis reveals that BnSOS2 is salt-inducible. Our results indicate that BnSOS2 is a new member of the plant SOS2 gene family, which may play an important role in salt tolerance of plants.     

Purification and characterization of manganese-dependent alkaline serine protease from Bacillus pumilus TMS55

The purification and characterization of a Mn2+-dependent alkaline serine protease produced by Bacillus pumilus TMS55 were investigated. The enzyme was purified in three steps: concentrating the crude enzyme using ammonium sulfate precipitation, followed by gel filtration and cation-exchange chromatography. The purified protease had a molecular mass of approximately 35 kDa, was highly active over a broad pH range of 7.0 to 12.0, and remained stable over a pH range of 7.5 to 11.5. The optimum temperature for the enzyme activity was found to be 60 degreesC. PMSF and AEBSF (1 mM) significantly inhibited the protease activity, indicating that the protease is a serine protease. Mn2+ ions enhanced the activity and stability of the enzyme. In addition, the purified protease remained stable with oxidants (H2O2, 2%) and organic solvents (25%), such as benzene, hexane, and toluene. Therefore, these characteristics of the protease and its dehairing ability indicate its potential for a wide range of commercial applications.     

A deduced Thermomonospora curvata protein containing serine/threonine protein kinase and WD-repeat domains.

The gene pkwA coding for a typical WD-repeat protein was found in the chromosome of the bacterium Thermomonospora curvata CCM 3352. Until now WD-repeat proteins were through to be confined to eukaryotes.     

Cloning, overexpression, and characterization of a serine/threonine protein kinase pknI from Mycobacterium tuberculosis H37Rv

Protein phosphorylation-dephosphorylation is the principal mechanism for translation of external signals into cellular responses. Eukaryotic-like serine/threonine kinases have been reported to play important roles in bacterial development and/or virulence. The PknI protein is one of the 11 eukaryotic-like serine/threonine kinases in Mycobacterium tuberculosis H37Rv. From the bioinformatic studies, PknI protein has been shown to have an N-terminal cytoplasmic domain followed by a transmembrane region and an extracellular C-terminus suggestive of a sensor molecule. In this study, we have cloned, overexpressed, and characterized the entire coding region and the cytoplasmic domain of PknI as a fusion protein with an N-terminal histidine tag, and used immobilized metal affinity chromatography for purification of recombinant proteins. The purified recombinant proteins were found to be functionally active through in vitro phosphorylation assay and phosphoamino acid analysis. In vitro kinase assay of both proteins revealed that PknI is capable of autophosphorylation and showed manganese-dependent activity. Phosphoamino acid analysis indicated phosphorylation at serine and threonine residues. Southern blot analysis with genomic DNA highlighted the conserved nature of pknI among the various mycobacterial species. In silico analysis revealed a close homology of PknI to Stk1 from Streptococcus agalactiae, shown to have a role in virulence and cell segregation of the organism.     

Biochemical characterization of a family of serine/threonine protein kinases regulated by tyrosine and serine/threonine phosphorylations.

Mitogen-activated protein kinase (p42mapk) becomes transiently activated after treatment of serum-starved murine Swiss 3T3 cells or EL4 thymocytes with a diversity of mitogens. Similarly, a meiosis-activated protein kinase (p44mpk) becomes stimulated during maturation of sea star oocytes induced by 1-methyladenine. Both p42mapk and p44mpk have been identified as protein-serine/threonine kinases that are activated as a consequence of their phosphorylation. Because homologous protein kinases may play essential roles in both mitogenesis and oogenesis, we have compared in detail the biochemical properties of these two kinases. We find that these kinases are highly related based on their in vitro substrate specificities, sensitivity to inhibitors, and immunological cross-reactivity. However, they differ in apparent molecular weight and can be separated chromatographically, indicating that the two enzymes are distinct. Furthermore, in the course of this investigation, we have identified a 44-kDa protein kinase in mitogen-stimulated Swiss mouse 3T3 cells and EL4 thymocytes that co-purifies with p44mpk and thus appears to be a closer homolog of the sea star enzyme. Analysis of these protein kinases clarifies the relationships between a set of tyrosine-phosphorylated 41-45-kDa proteins present in mitogen-stimulated cells (Martinez, R., Nakamura., K. D., and Weber, M. J. (1982) Mol. Cell. Biol. 2, 653-655; Cooper, J. A., and Hunter, T. (1984) Mol. Cell. Biol. 4, 30-37), two myelin basic protein kinases identified in epidermal growth factor-treated Swiss mouse 3T3 cells (Ahn, N. G., Weiel, J. E., Chan, C. P., and Krebs, E. G. (1990) J. Biol. Chem. 265, 11487-11494), and p42mapk. Our work points to the existence of a group of related serine/threonine protein kinases, regulated by tyrosine phosphorylation and functioning at different stages of the cell cycle.     

Molecular cloning and characterization of the human protein kinase D2. A novel member of the protein kinase D family of serine threonine kinases

We have isolated the full-length cDNA of a novel human serine threonine protein kinase gene. The deduced protein sequence contains two cysteine-rich motifs at the N terminus, a pleckstrin homology domain, and a catalytic domain containing all the characteristic sequence motifs of serine protein kinases. It exhibits the strongest homology to the serine threonine protein kinases PKD/PKCmicro and PKCnu, particularly in the duplex zinc finger-like cysteine-rich motif, in the pleckstrin homology domain and in the protein kinase domain. In contrast, it shows only a low degree of sequence similarity to other members of the PKC family. Therefore, the new protein has been termed protein kinase D2 (PKD2). The mRNA of PKD2 is widely expressed in human and murine tissues. It encodes a protein with a molecular mass of 105 kDa in SDS-polyacrylamide gel electrophoresis, which is expressed in various human cell lines, including HL60 cells, which do not express PKCmicro. In vivo phorbol ester binding studies demonstrated a concentration-dependent binding of [(3)H]phorbol 12,13-dibutyrate to PKD2. The addition of phorbol 12,13-dibutyrate in the presence of dioleoylphosphatidylserine stimulated the autophosphorylation of PKD2 in a synergistic fashion. Phorbol esters also stimulated autophosphorylation of PKD2 in intact cells. PKD2 activated by phorbol esters efficiently phosphorylated the exogenous substrate histone H1. In addition, we could identify the C-terminal Ser(876) residue as an in vivo phosphorylation site within PKD2. Phosphorylation of Ser(876) of PKD2 correlated with the activation status of the kinase. Finally, gastrin was found to be a physiological activator of PKD2 in human AGS-B cells stably transfected with the CCK(B)/gastrin receptor. Thus, PKD2 is a novel phorbol ester- and growth factor-stimulated protein kinase.     

Identification, purification, and characterization of a novel serine/threonine protein phosphatase from bovine brain.

A novel serine/threonine protein phosphatase is identified, and the catalytic subunit, obtained from a detergent extraction of the pellet generated by a 100,000 x g centrifugation of a whole bovine brain homogenate, is purified and characterized. The protein phosphatase, designated as PP3, has a Mr of 36,000, does not require divalent cations for activity, is stimulated rather than inhibited by inhibitor 2, is inhibited by both okadaic acid and microcystin-LR with an intermediate IC50 compared to type 1 and type 2A protein phosphatases, and preferentially dephosphorylates the beta subunit of phosphorylase kinase. Substrate specificity, immunoblotting with type-specific antisera, and the amino acid sequences of peptides derived from PP3 indicate that PP3 is not an isoform of any known serine/threonine protein phosphatase.     

Adenovirus E1A is associated with a serine/threonine protein kinase.

The adenovirus E1A proteins form stable protein complexes with a number of cellular proteins, including cyclin A and the product of the retinoblastoma susceptibility gene. We have been interested in learning about the function of proteins associated with E1A and therefore looked for an enzymatic activity present in E1A complexes. We found a serine/threonine kinase activity that phosphorylates two proteins bound to E1A, the 107- and 130-kDa (107K and 130K) proteins. The kinase also phosphorylates histone H1 added as an exogenous substrate. The kinase activity is cell cycle regulated, being most active in S and G2/M-phase cells. The timing of phosphorylation of the 107K protein in vitro correlates with the phosphorylation pattern of the 107K protein in vivo. A variety of genetic and immunochemical approaches indicate that the activity is probably not due to the E1A-associated 300K, 130K, 107K, or pRB protein. Although we have not established the identity of the kinase, we present evidence that the kinase activity is consistent with phosphorylation by p34cdc2 or a related kinase.     

A tightly associated serine/threonine protein kinase regulates phosphoinositide 3-kinase activity.

We identified a serine/threonine protein kinase that is associated with and phosphorylates phosphoinositide 3-kinase (PtdIns 3-kinase). The serine kinase phosphorylates both the 85- and 110-kDa subunits of PtdIns 3-kinase and purifies with it from rat liver and immunoprecipitates with antibodies raised to the 85-kDa subunit. Tryptic phosphopeptide maps indicate that p85 from polyomavirus middle T-transformed cells is phosphorylated in vivo at three sites phosphorylated in vitro by the associated serine kinase. The 85-kDa subunit of PtdIns 3-kinase is phosphorylated in vitro on serine at a stoichiometry of approximately 1 mol of phosphate per mol of p85. This phosphorylation results in a three- to sevenfold decrease in PtdIns 3-kinase activity. Dephosphorylation with protein phosphatase 2A reverses the inhibition. This suggests that the association of protein phosphatase 2A with middle T antigen may function to activate PtdIns 3-kinase.     

Activation of a manganese-dependent membrane protein kinase by serine and tyrosine phosphorylation.

A Mn2(+)-dependent serine/threonine protein kinase from rat liver membranes copurifies with the insulin receptor (IR) on wheat germ agglutinin (WGA)-sepharose. The kinase is present in a nonactivated form in membranes but can be activated 20-fold by phosphorylating the WGA-sepharose fraction with casein kinase-1 (CK-1), casein kinase-2 (CK-2), or casein kinase-3 (CK-3). The activated kinase can use IR beta-subunit, myelin basic protein, and histones as substrates. Activation of the kinase seems to proceed by two or more steps. Sodium vanadate and Mn2+ are required in reaction mixtures for activation to be observed, whereas the tyrosine kinase-specific substrate, poly (glu, tyr), completely inhibits activation. These observations suggest that, in addition to serine/threonine phosphorylation by one of the casein kinases, activation of the Mn2(+)-dependent protein kinase also requires tyrosine phosphorylation. Such phosphorylation may be catalyzed by the IR tyrosine kinase.     

Brain protein serine/threonine phosphatases.

All of the known protein serine/threonine phosphatases are expressed in the brain. These enzymes participate in a variety of signaling pathways that modulate neuronal activity. The multifunctional activity of many serine/threonine phosphatases is achieved through their association with targeting proteins. Identification and analysis of targeting molecules has led to new insights into the functions of protein phosphatases in neuronal signaling. The recent use of transgenic mice has also increased our understanding of the physiological roles of these enzymes in the brain.     

A developmentally regulated, putative serine/threonine protein kinase is essential for development in Dictyostelium.

Using PCR technology, we have cloned parts of three developmentally regulated putative serine/threonine kinases from Dictyostelium. All show significant homology to members of the cAMP-dependent protein kinase A/protein kinase C subfamilies. A genomic clone encoding one of these, DdPK3, has been isolated and sequenced. The open reading frame encodes a protein of 648 amino acids with the conserved kinase domain in the C-terminal half. The protein encoded by this gene is unusual in that it contains long homopolymer runs in the N-terminal half of the protein, including a long run of 88 amino acids in which 73 are glutamine residues. To examine the function of DdPK3, a gene disruption was created via homologous recombination. Ddpk3- cells do not aggregate by themselves but will co-aggregate with wild-type cells. However, after aggregation these cells are 'sloughed off' and do not proceed further through development, but are found as a discrete mass alongside the fruiting body formed by the wild-type cells. Analysis of signal transduction pathways indicates that cAMP pulse-induced expression of aggregation stage-specific genes is normal in Ddpk3- cells, as is induction of the prestalk gene Ddras in single cell assays. However, cAMP induction of the late promoters of cAMP receptor cAR1 and of two prespore-specific genes is absent under similar conditions. These cells show normal activation of adenylate cyclase and normal phosphorylation of the G alpha protein G alpha 2 in response to cAMP. The possible role of DdPK3 in Dictyostelium development is discussed.     

Isolation and characterization of activin receptor from mouse embryonal carcinoma cells. Identification of its serine/threonine/tyrosine protein kinase activity.

The activin receptor protein was isolated from the mouse embryonal carcinoma (EC) cell line P19 by three cycles of affinity chromatography on an activin A-immobilized column. The purified receptor had a specific and high affinity for activins A, AB, and B (Kd = 345 pM), but not for transforming growth factor beta. The purified activin receptor was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and ligand blotting analysis as a single protein of 70 kDa. The amino acid sequence of the first 18 NH2-terminal residues revealed that the receptor is a member of the activin receptor family. The purified receptor phosphorylated itself and exogenous substrate proteins on serine, threonine, and tyrosine residues, indicating that the activin receptor is a transmembrane serine/threonine/tyrosine protein kinase. These results suggest that signal transduction of activin employs a novel pathway via a new class of cellular receptor in EC P19 cells.