A eukaryotic-type serine/threonine protein kinase StkP of Streptococcus pneumoniae acts as a dimer in vivo

Streptococcus pneumoniae carries a single Ser/Thr protein kinase gene stkP in its genome. Biochemical studies performed with recombinant StkP have revealed that this protein is a functional membrane-linked eukaryotic-type Ser/Thr protein kinase. Here, we demonstrate that the deletion of its extracellular domain negatively affects the stability of a core kinase domain. In contrast, the membrane anchored kinase domain and the full-length form of StkP were stable and capable of autophosphorylation. Furthermore, evidence is presented that StkP forms dimers through its transmembrane and extracellular domains.     

Phosphorylation of protein phosphatase inhibitor-1 by protein kinase C

Inhibitor-1 becomes a potent inhibitor of protein phosphatase 1 when phosphorylated by cAMP-dependent protein kinase at Thr(35). Moreover, Ser(67) of inhibitor-1 serves as a substrate for cyclin-dependent kinase 5 in the brain. Here, we report that dephosphoinhibitor-1 but not phospho-Ser(67) inhibitor-1 was efficiently phosphorylated by protein kinase C at Ser(65) in vitro. In contrast, Ser(67) phosphorylation by cyclin-dependent kinase 5 was unaffected by phospho-Ser(65). Protein kinase C activation in striatal tissue resulted in the concomitant phosphorylation of inhibitor-1 at Ser(65) and Ser(67), but not Ser(65) alone. Selective pharmacological inhibition of protein phosphatase activity suggested that phospho-Ser(65) inhibitor-1 is dephosphorylated by protein phosphatase 1 in the striatum. In vitro studies confirmed these findings and suggested that phospho-Ser(67) protects phospho-Ser(65) inhibitor-1 from dephosphorylation by protein phosphatase 1 in vivo. Activation of group I metabotropic glutamate receptors resulted in the up-regulation of diphospho-Ser(65)/Ser(67) inhibitor-1 in this tissue. In contrast, the activation of N-methyl-d-aspartate-type ionotropic glutamate receptors opposed increases in striatal diphospho-Ser(65)/Ser(67) inhibitor-1 levels. Phosphomimetic mutation of Ser(65) and/or Ser(67) did not convert inhibitor-1 into a protein phosphatase 1 inhibitor. On the other hand, in vitro and in vivo studies suggested that diphospho-Ser(65)/Ser(67) inhibitor-1 is a poor substrate for cAMP-dependent protein kinase. These observations extend earlier studies regarding the function of phospho-Ser(67) and underscore the possibility that phosphorylation in this region of inhibitor-1 by multiple protein kinases may serve as an integrative signaling mechanism that governs the responsiveness of inhibitor-1 to cAMP-dependent protein kinase activation.     

Cell Wall Stress Stimulates the Activity of the Protein Kinase StkP of Streptococcus pneumoniae,Leading to Multiple Phosphorylation

Streptococcus pneumoniae is an opportunistic human pathogen that encodes a single eukaryotic-type Ser/Thr protein kinase StkP and its functional counterpart, the protein phosphatase PhpP. These signaling enzymes play critical roles in coordinating cell division and growth in pneumococci. In this study, we determined the proteome and phosphoproteome profiles of relevant mutants. Comparison of those with the wild-type provided a representative dataset of novel phosphoacceptor sites and StkP-dependent substrates. StkP phosphorylates key proteins involved in cell division and cell wall biosynthesis in both the unencapsulated laboratory strain Rx1 and the encapsulated virulent strain D39. Furthermore, we show that StkP plays an important role in triggering an adaptive response induced by a cell wall-directed antibiotic. Phosphorylation of the sensor histidine kinase WalK and downregulation of proteins of the WalRK core regulon suggest crosstalk between StkP and the WalRK two-component system. Analysis of proteomic profiles led to the identification of gene clusters regulated by catabolite control mechanisms, indicating a tight coupling of carbon metabolism and cell wall homeostasis. The imbalance of steady-state protein phosphorylation in the mutants as well as after antibiotic treatment is accompanied by an accumulation of the global Spx regulator, indicating a Spx-mediated envelope stress response. In summary, StkP relays the perceived signal of cell wall status to key cell division and regulatory proteins, controlling the cell cycle and cell wall homeostasis.     

Comparison of phosphorylation sites in protamines between protein kinase C and cAMP-dependent protein kinase

Phosphorylation sites of protamines by protein kinase C and cAMP-dependent protein kinase (protein kinase A) were studied. Using clupeine Y1 as a substrate, protein kinase C phosphorylates both Ser and Thr residues, whereas protein kinase A phosphorylates only Ser residue(s). Protein kinase C phosphorylates all Ser and Thr residues of clupeine Y2 and Z, however protein kinase A phosphorylates mainly Ser9 and slightly Thr5 in clupeine Y2 and Ser6 and Ser10 in clupeine Z. These results suggest that protein kinase C recognizes more sites than those of protein kinase A and may participate in protamine phosphorylation in vivo.     

Functional interaction between the Ser/Thr kinase PKL12 and N-acetylglucosamine kinase, a prominent enzyme implicated in the salvage pathway for GlcNAc recycling.

PKL12 (STK16) is a ubiquitously expressed Ser/Thr kinase, not structurally related to the well known subfamilies, with a putative role in cell adhesion control. Yeast two-hybrid protein interaction screening was used to search for proteins that associate with PKL12 and to delineate signaling pathways and/or regulatory circuits in which this kinase participates. One positive clone contained an open reading frame highly similar to N-acetylglucosamine kinase (GlcNAcK) of several species. The PKL12/GlcNAcK interaction was further confirmed both in vitro and in vivo. Protein expression analysis of GlcNAcK using a specific rabbit antiserum displayed a ubiquitous pattern in cell lines and animal tissues. Subcellular localization studies showed that GlcNAcK is a cytoplasmic protein with a dual subcellular localization, distributed between the perinuclear and peripheral cell reservoirs. After overexpression, GlcNAcK localizes in vesicular structures associated mainly with the cell membrane and colocalizes with the PKL12 protein. GlcNAcK is not otherwise a substrate for PKL12 activity and PKL12 does not appear to influence GlcNAcK activity either in vitro or in vivo. In vitro kinase assays have nonetheless revealed that functional GlcNAcK, although not able to modulate autophosphorylation of PKL12, greatly influences PKL12 kinase activity on a defined substrate protein. These results are interpreted to indicate a potential in vivo role for GlcNAcK in PKL12 translocation and a tentative regulatory role for PKL12-mediated phosphorylation on substrate proteins.     

PknB kinase activity is regulated by phosphorylation in two Thr residues and dephosphorylation by PstP,the cognate phospho-Ser/Thr phosphatase,in Mycobacterium tuberculosis

Bacterial genomics revealed the widespread presence of eukaryotic-like protein kinases and phosphatases in prokaryotes, but little is known on their biochemical properties, regulation mechanisms and physiological roles. Here we focus on the catalytic domains of two trans-membrane enzymes, the Ser/Thr protein kinase PknB and the protein phosphatase PstP from Mycobacterium tuberculosis. PstP was found to specifically dephosphorylate model phospho-Ser/Thr substrates in a Mn2+-dependent manner. Autophosphorylated PknB was shown to be a substrate for Pstp and its kinase activity was affected by PstP-mediated dephosphorylation. Two threonine residues in the PknB activation loop, found to be mostly disordered in the crystal structure of this kinase, namely Thr171 and Thr173, were identified as the target for PknB autophosphorylation and PstP dephosphorylation. Replacement of these threonine residues by alanine significantly decreased the kinase activity, confirming their direct regulatory role. These results indicate that, as for eukaryotic homologues, phosphorylation of the activation loop provides a regulation mechanism of mycobacterial kinases and strongly suggest that PknB and PstP could work as a functional pair in vivo to control mycobacterial cell growth.     

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.     

pp54 microtubule-associated protein 2 kinase. A novel serine/threonine protein kinase regulated by phosphorylation and stimulated by poly-L-lysine

An hepatic protein kinase that phosphorylates microtubule-associated protein 2 (MAP-2) on Ser/Thr residues is markedly activated after intraperitoneal injection of cycloheximide in the rat. The enzyme has been purified greater than 10,000-fold to near homogeneity and corresponds to a 54-kDa polypeptide, based on auto-phosphorylation, renaturation of activity from sodium dodecyl sulfate gels, and gel filtration. The protein kinase activity is unaffected by prior autophosphorylation, Ca2+, diacylglycerol and phospholipids, cyclic nucleotides, staurosporine, and protein kinase inhibitor, but can be totally and specifically deactivated by the Ser/Thr protein phosphatase 2A. The enzyme is inhibited completely but reversible by transition metals and p-chloromercuribenzoate, and is strongly stimulated by poly-L-lysine toward most, but not all protein substrates. The activity of the cycloheximide-stimulated MAP-2 kinase (pp54 MAP-2 kinase) toward potential polypeptide substrates was compared to that of an insulin-stimulated MAP-2 kinase (pp42 MAP-2 kinase). Although both MAP-2 kinases exhibited little or no ability to phosphorylate histones and casein, the two kinases had a distinguishable substrate specificity. At comparable MAP-2 phosphorylating activities, pp42 MAP-2 kinase, but not pp54 MAP-2 kinase, phosphorylated and activated the Xenopus S6 protein kinase II. Moreover, pp42 MAP-2 kinase phosphorylated myelin basic protein at 10-12-fold higher rates than did pp54 MAP-2 kinase. Cycloheximide-activated pp54 MAP-2 protein kinase appears to be a previously uncharacterized protein kinase that is itself regulated through Ser/Thr phosphorylation and, perhaps, polypeptide regulators with basic domains. The identity of the upstream regulatory elements and the native substrates remain to be established.     

Phosphorylation of protein phosphatase 2Czeta by c-Jun NH2-terminal kinase at Ser92 attenuates its phosphatase activity

The protein phosphatase 2C (PP2C) family represents one of the four major protein Ser/Thr phosphatase activities in mammalian cells and contains at least 13 distinct gene products. Although PP2C family members regulate a variety of cellular functions, mechanisms of regulation of their activities are largely unknown. Here, we show that PP2Czeta, a PP2C family member that is enriched in testicular germ cells, is phosphorylated by c-Jun NH 2-terminal kinase (JNK) but not by p38 in vitro. Mass spectrometry and mutational analyses demonstrated that phosphorylation occurs at Ser (92), Thr (202), and Thr (205) of PP2Czeta. Phosphorylation of these Ser and Thr residues of PP2Czeta ectopically expressed in 293 cells was enhanced by osmotic stress and was attenuated by a JNK inhibitor but not by p38 or MEK inhibitors. Phosphorylation of PP2Czeta by TAK1-activated JNK repressed its phosphatase activity in cells, and alanine mutation at Ser (92) but not at Thr (202) or Thr (205) suppressed this inhibition. Taken together, these results suggest that specific phosphorylation of PP2Czeta at Ser (92) by stress-activated JNK attenuates its phosphatase activity in cells.     

Mutational dissection of the S/T-kinase StkP reveals crucial roles in cell division of Streptococcus pneumoniae

Eukaryotic-like serine/threonine-kinases are involved in the regulation of a variety of physiological processes in bacteria. In Streptococcus pneumoniae, deletion of the single serine/threonine-kinase gene stkP results in an aberrant cell morphology suggesting that StkP participates in pneumococcus cell division. To understand the function of StkP, we have engineered various pneumococcus strains expressing truncated or kinase-dead forms of StkP. We show that StkP kinase activity, but also its extracellular and cytoplasmic domains per se, are required for pneumococcus cell division. Indeed, we observe that mutant cells show round or elongated shapes with non-functional septa and a chain phenotype, delocalized sites of peptidoglycan synthesis and diffused membrane StkP localization. To gain understanding of the underlying StkP-mediated regulatory mechanism, we show that StkP specifically phosphorylates in vivo the cell division protein DivIVA on threonine 201. Pneumococcus cells expressing non-phosphorylatable DivIVA-T201A possess an elongated shape with a polar bulge and aberrant spatial organization of nascent peptidoglycan. This brings the first evidence of the importance of StkP in relationship to the phosphorylation of one of its substrates in cell division. It is concluded that StkP is a multifunctional protein that plays crucial functions in pneumococcus cell shape and division.     

Regulation of NDR protein kinase by hydrophobic motif phosphorylation mediated by the mammalian Ste20-like kinase MST3

NDR protein kinases are involved in the regulation of cell cycle progression and morphology. NDR1/NDR2 protein kinase is activated by phosphorylation on the activation loop phosphorylation site Ser281/Ser282 and the hydrophobic motif phosphorylation site Thr444/Thr442. Autophosphorylation of NDR is responsible for phosphorylation on Ser281/Ser282, whereas Thr444/Thr442 is targeted by an upstream kinase. Here we show that MST3, a mammalian Ste20-like protein kinase, is able to phosphorylate NDR protein kinase at Thr444/Thr442. In vitro, MST3 selectively phosphorylated Thr442 of NDR2, resulting in a 10-fold stimulation of NDR activity. MOB1A (Mps one binder 1A) protein further increased the activity, leading to a fully active kinase. In vivo, Thr442 phosphorylation after okadaic acid stimulation was potently inhibited by MST3KR, a kinase-dead mutant of MST3. Knockdown of MST3 using short hairpin constructs abolished Thr442 hydrophobic motif phosphorylation of NDR in HEK293F cells. We conclude that activation of NDR is a multistep process involving phosphorylation of the hydrophobic motif site Thr444/2 by MST3, autophosphorylation of Ser281/2, and binding of MOB1A.     

Regulation of Ca(2+)/calmodulin-dependent protein kinase kinase alpha by cAMP-dependent protein kinase: I. Biochemical analysis

Ca(2+)/calmodulin-dependent protein kinases (CaM-kinases) I and IV are activated upon phosphorylation of their Thr(177) and Thr(196), respectively, by the upstream Ca(2+)/calmodulin-dependent protein kinases CaM-kinase kinase alpha and beta, and deactivated upon dephosphorylation by protein phosphatases such as CaM-kinase phosphatase. Recent studies demonstrated that the activity of CaM-kinase kinase alpha is decreased upon phosphorylation by cAMP-dependent protein kinase (PKA), and the relationship between the inhibition and phosphorylation of CaM-kinase kinase alpha by PKA has been studied. In the present study, we demonstrate that the activity of CaM-kinase kinase alpha toward PKIV peptide, which contains the sequence surrounding Thr(196) of CaM-kinase IV, is increased by incubation with PKA in the presence of Ca(2+)/calmodulin but decreased in its absence, while the activity toward CaM-kinase IV is decreased by incubation with PKA in both the presence and absence of Ca(2+)/calmodulin. Six phosphorylation sites on CaM-kinase kinase alpha, Ser(24) for autophosphorylation, and Ser(52), Ser(74), Thr(108), Ser(458), and Ser(475) for phosphorylation by PKA, were identified by amino acid sequence analysis of the phosphopeptides purified from the tryptic digest of the phosphorylated enzymes. The presence of Ca(2+)/calmodulin suppresses phosphorylation on Ser(52), Ser(74), Thr(108), and Ser(458) by PKA, but accelerates phosphorylation on Ser(475). The changes in the activity of the enzyme upon phosphorylation appear to occur as a result of conformational changes induced by phosphorylation on several sites.     

Regulation of protein phosphatase inhibitor-1 by cyclin-dependent kinase 5

Inhibitor-1, the first identified endogenous inhibitor of protein phosphatase 1 (PP-1), was previously reported to be a substrate for cyclin-dependent kinase 5 (Cdk5) at Ser67. Further investigation has revealed the presence of an additional Cdk5 site identified by mass spectrometry and confirmed by site-directed mutagenesis as Ser6. Basal levels of phospho-Ser6 inhibitor-1, as detected by a phosphorylation state-specific antibody against the site, existed in specific regions of the brain and varied with age. In the striatum, basal in vivo phosphorylation and dephosphorylation of Ser6 were mediated by Cdk5, PP-2A, and PP-1, respectively. Additionally, calcineurin contributed to dephosphorylation under conditions of high Ca2+. In biochemical assays the function of Cdk5-dependent phosphorylation of inhibitor-1 at Ser6 and Ser67 was demonstrated to be an intramolecular impairment of the ability of inhibitor-1 to be dephosphorylated at Thr35; this effect was recapitulated in two systems in vivo. Dephosphorylation of inhibitor-1 at Thr35 is equivalent to inactivation of the protein, as inhibitor-1 only serves as an inhibitor of PP-1 when phosphorylated by cAMP-dependent kinase (PKA) at Thr35. Thus, inhibitor-1 serves as a critical junction between kinase- and phosphatase-signaling pathways, linking PP-1 to not only PKA and calcineurin but also Cdk5.     

Mechanism of activation of protein kinase B by insulin and IGF-1.

Insulin activated endogenous protein kinase B alpha (also known as RAC/Akt kinase) activity 12-fold in L6 myotubes, while after transfection into 293 cells PKBalpha was activated 20- and 50-fold in response to insulin and IGF-1 respectively. In both cells, the activation of PKBalpha was accompanied by its phosphorylation at Thr308 and Ser473 and, like activation, phosphorylation of both of these residues was prevented by the phosphatidylinositol 3-kinase inhibitor wortmannin. Thr308 and/or Ser473 were mutated to Ala or Asp and activities of mutant PKBalpha molecules were analysed after transfection into 293 cells. The activity of wild-type and mutant PKBalpha was also measured in vitro after stoichiometric phosphorylation of Ser473 by MAPKAP kinase-2. These experiments demonstrated that activation of PKBalpha by insulin or insulin-like growth factor-1 (IGF-1) results from phosphorylation of both Thr308 and Ser473, that phosphorylation of both residues is critical to generate a high level of PKBalpha activity and that the phosphorylation of Thr308 in vivo is not dependent on phosphorylation of Ser473 or vice versa. We propose a model whereby PKBalpha becomes phosphorylated and activated in insulin/IGF-1-stimulated cells by an upstream kinase(s).     

Relationship between ATM and ribosomal protein S6 revealed by the chemical inhibition of Ser/Thr protein phosphatase type 1

The optimal cellular responses to DNA damage are modulated by kinase and phosphatase. The ataxia telangiectasia mutated (ATM) is a Ser/Thr kinase which is the core of the DNA damage signaling apparatus. The Ser/Thr protein phosphatase type 1 (PP1) inhibitor, tautomycetin (TC) and an antibody to the phospho-(S/T)Q sites of the ATM substrate were used to identify the common substrates for PP1 and ATM in regulating the pathway for DNA damage response. Ribosomal protein S6 (RPS6) was first identified as a substrate for PP1 and ATM. The phosphorylation at Ser247 of RPS6 was then significantly decreased by PP1-mediated dephosphorylation immediately after UV irradiation. These results suggest that PP1 specifically dephosphorylated RPS6 at phospho-Ser247 in vivo. In response to DNA damage, ATM activity was finally required for the phosphorylation of RPS6 at Ser247. We propose from these results a novel mechanism for modulating the RPS6 function by PP1 and ATM which regulates cell growth and survival in response to DNA-damage stimuli.     

Plasmodium falciparum protein phosphatase type 1 functionally complements a glc7 mutant in Saccharomyces cerevisiae

We have identified a new homologue of protein phosphatase type 1 from Plasmodium falciparum, designated PfPP1, which shows 83-87% sequence identity with yeast and mammalian PP1s at the amino acid level. The PfPP1 sequence is strikingly different from all other P. falciparum Ser/Thr phosphatases cloned so far. The deduced 304 amino acid sequence revealed the signature sequence of Ser/Thr phosphatase LRGNHE, and two putative protein kinase C and five putative casein kinase II phosphorylation sites. Calyculin A, a potent inhibitor of Ser/Thr phosphatase 1 and 2A showed hyperphosphorylation of a 51kDa protein among other parasite proteins. Okadaic acid on the other hand, was without any effect suggesting that PP1 activity might predominate over PP2A activity in intra-erythrocytic P. falciparum. Complementation studies showed that PfPP1 could rescue low glycogen phenotype of Saccharomyces cerevisiae glc7 (PP1) mutant, strongly suggesting functional interaction of PfPP1 and yeast proteins involved in glycogen metabolism.     

The sole Serine/Threonine protein kinase and its cognate phosphatase from Aquifex aeolicus targets Pyrimidine biosynthesis

Serine/Threonine kinases participate in complex, interacting signaling pathways in eukaryotes, prokaryotes, and archae. While most organisms contain many different kinases, the extreme hyperthermophile, Aquifex aeolicus encodes a single hypothetical Ser/Thr kinase. A gene homologous to eukaryotic protein phosphatases overlaps the kinase gene by a single base pair. The putative kinase, AaSTPK and phosphatase, AaPPM, were cloned and expressed in E. coli, purified to homogeneity and found to be functional. AaSTPK is a 34-kDa monomer that can use MgATP, MnATP, or MnGTP as co-substrates, although MgATP appears to be the preferred substrate. AaSTPK was autophosphorylated on a threonine residue and was dephosphorylated by AaPPM. AaPPM phosphatase is homologous to the PPM sub-family of Ser/Thr phosphatases and was stimulated by MnCl₂ and CoCl₂ but not MgCl₂. AaSTPK also phosphorylated one threonine residue on the carbamoyl phosphate synthetase, CPS.A subunit. Carbamoyl phosphate synthetase reconstituted with phosphorylated CPS.A had unaltered catalytic activity but allosteric inhibition by UMP and activation by the arginine intermediate, ornithine, were both appreciably attenuated. These changes in allosteric regulation would be expected to activate pyrimidine biosynthesis by releasing the constraints imposed on carbamoyl phosphate synthetase activity by UMP and uncoupling the regulation of pyrimidine and arginine biosynthesis. CPS.A was also dephosphorylated by AaPPM. Aquifex aeolicus occupies the lowest branch on the prokaryotic phylogenetic tree. The Thr/Ser kinase, its cognate phosphatase and a protein substrate may be elements of a simple signaling pathway, perhaps the most primitive example of this mode of regulation described thus far.