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.     

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.     

Multiple phosphorylation sites in the β subunit of thylakoid ATP synthase

Proteomic analyses of the β subunit of the plastid ATP synthase of barley (Hordeum vulgare L.) revealed that mature protein was not carboxy terminus processed and suggested the correction of the 274 codon (GAT to AAT) in the data bank that was confirmed by DNA sequencing. Six isoforms of the ATP synthase β subunit with pI ranging from 4.95 to 5.14 were resolved by two-dimensional electrophoresis (2-DE). Mass spectrometry analyses indicated that the six isoforms differ in their phosphorylation degree, which was confirmed by the disappearance of more acidic forms after incubation with the protein phosphatase calcineurin. Six Ser and/or Thr were detected as phosphorylated, among them the conserved Thr-179 that is also phosphorylated in the β subunit of human mitochondria. The results are discussed in relation with the proposed regulation of the ATP synthase by phosphorylation and 14-3-3 proteins.     

Protein kinase associated with ribosomes of streptomycetes

Protein kinases can be classified into two main superfamilies on the basis of their sequence similarity and substrate specificity. The protein His kinase superfamily which autophosphorylate a His residue, and superfamily Ser/Thr and Tyr protein kinases, which phosphorylate Ser, Thr or Tyr residues. During the last years genes encoding Ser/Thr protein kinases have been identified in several microorganisms. Phosphorylation of proteins on Ser/Thr residues can be involved in many functions of prokaryotic cells including cell differentiation, signal transduction and protein biosynthesis. Phosphorylation of prokaryotic protein-synthesizing systems showed that the phosphorylation of initiation and elongation factors is subject to alteration during cell differentiation or bacteriophage infection. Protein kinase associated with ribosomes of streptomycetes phosphorylate the elongation factor Tu and 11 ribosomal proteins even in bacteriophage-uninfected cells. After phosphorylation of ribosomal proteins, ribosomes lose about 30% of their activity at the translation of poly(U). Presented at theSymposium on Regulation of Translation of Genetic Information by Protein Phosphorylation, 21st Congress of the Czechoslovak Society for Microbiology, Hradec Králové (Czech Republic), September 6–10, 1998.     

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 Streptococcus pyogenes orphan protein tyrosine phosphatase,SP‐PTP,possesses dual specificity and essential virulence regulatory functions

Group A Streptococcus (GAS) is a human pathogen that causes high morbidity and mortality. GAS lacks a gene encoding tyrosine kinase but contains one encoding tyrosine phosphatase (SP‐PTP). Thus, GAS is thought to lack tyrosine phosphorylation, and the physiological significance of SP‐PTP is, therefore, questionable. Here, we demonstrate that SP‐PTP possesses dual phosphatase specificity for Tyr‐ and Ser/Thr‐phosphorylated GAS proteins, such as Ser/Thr kinase (SP‐STK) and the SP‐STK‐phosphorylated CovR and WalR proteins. Phenotypic analysis of GAS mutants lacking SP‐PTP revealed that the phosphatase activity per se positively regulates growth, cell division and the ability to adhere to and invade host cells. Furthermore, A549 human lung cells infected with GAS mutants lacking SP‐PTP displayed increased Ser‐/Thr‐/Tyr‐phosphorylation. SP‐PTP also differentially regulates the expression of ～50% of the total GAS genes, including several virulence genes potentially through the two‐component regulators, CovR, WalR and PTS/HPr regulation of Mga. Although these mutants exhibit attenuated virulence, a GAS mutant overexpressing SP‐PTP is hypervirulent. Our study provides the first definitive evidence for the presence and importance of Tyr‐phosphorylation in GAS and the relevance of SP‐PTP as an important therapeutic target.     

p21-activated kinase 2 in neutrophils can be regulated by phosphorylation at multiple sites and by a variety of protein phosphatases

The p21-activated kinase(Pak) 2 undergoes rapid autophosphorylation/activation in neutrophils stimulated with a variety of chemoattractants (e.g., fMLP). Phosphorylation within the activation loop (Thr(402)) and inhibitory domain (Ser(141)) is known to increase the activity of Pak in vitro, whereas phosphorylation within the Nck (Ser(20)) and Pak-interacting guanine nucleotide exchange factor (Ser(192) and Ser(197)) binding sites blocks the interactions of Pak 2 with these proteins. A panel of phosphospecific Abs was used to investigate the phosphorylation of Pak 2 in neutrophils at these sites. Pak 2 underwent rapid (< or =15 s) phosphorylation at Ser(20), Ser(192/197), and Thr(402) in neutrophils stimulated with fMLP. Phosphorylation at Ser(192/197) and Thr(402) were highly transient events, whereas that at Ser(20) was more persistent. In contrast, Pak 2 was constitutively phosphorylated at Ser(141) in unstimulated neutrophils and phosphorylation at this site was less sensitive to cell stimulation than at other residues. Studies with selective inhibitors suggested that a variety of phosphatases might be involved in the rapid dephosphorylation of Pak 2 at Thr(402) in stimulated neutrophils. This was consistent with biochemical studies which showed that the activation loop of GST-Pak 3, which is homologous to that in Pak 2, was a substrate for protein phosphatase 1, 2A, and a Mg(2+)/Mn(2+)-dependent phosphatase(s) which exhibited properties different from those of the conventional isoforms of protein phosphatase 2C. The data indicate that Pak 2 undergoes a complex pattern of phosphorylation in neutrophils and that dephosphorylation at certain sites may involve multiple protein phosphatases that exhibit distinct modes of regulation.     

Development-dependent changes in the tight DNA-protein complexes of barley on chromosome and gene level

Background  

The tightly bound to DNA proteins (TBPs) is a protein group that remains attached to DNA with covalent or non-covalent bonds after its deproteinisation. The functional role of this group is as yet not completely understood. The main goal of this study was to evaluate tissue specific changes in the TBP distribution in barley genes and chromosomes in different phases of shoot and seed development. We have: 1. investigated the TBP distribution along Amy32b and Bmy1 genes encoding low pI α-amylase A and endosperm specific β-amylase correspondingly using oligonucleotide DNA arrays; 2. characterized the polypeptide spectrum of TBP and proteins with affinity to TBP-associated DNA; 3. localized the distribution of DNA complexes with TBP (TBP-DNA) on barley 1H and 7H chromosomes using mapped markers; 4. compared the chromosomal distribution of TBP-DNA complexes to the distribution of the nuclear matrix attachment sites.     

Identification of Tau and MAP2 as novel substrates of Rho-kinase and myosin phosphatase

Rho-kinase and myosin phosphatase are implicated in the phosphorylation-state of myosin light chain downstream of Rho, which is thought to induce smooth muscle contraction and stress fibre formation in non-muscle cells. Here, we found that microtubule-associated proteins, Tau and MAP2, interacted with the myosin-binding subunit (MBS) of myosin phosphatase, and were the possible substrates of both Rho-kinase and myosin phosphatase. We determined the phosphorylation sites of Tau (Thr245, Thr377, Ser409) and MAP2 (Ser1796) by Rho-kinase. We also found that Rho-kinase phosphorylated Tau at Ser262 to some extent. Phosphorylation by Rho-kinase decreased the activity of Tau to promote microtubule assembly in vitro. Substitutions of Ala for Ser/Thr at the phosphorylation sites of Tau (Tau-AAA) did not affect the activity to promote microtubule assembly, while substitutions of Asp for Ser/Thr (Tau-DDD), which are expected to mimic the phosphorylation-state of Tau, slightly reduced the activity. When Tau, or mutated forms of Tau, were expressed in PC12 cells, followed by treatment with cytochalasin D, they promoted extension of the cell process in a cytochalasin-dependent manner. However, Tau-DDD showed the weaker activity in this capacity than wild-type Tau or Tau-AAA. These results suggest that the phosphorylation-state of these residues of Tau affects its activity both in vitro and in vivo. Thus, it is likely that the Rho-kinase/MBS pathway regulates not only the actin-myosin system but also microtubule dynamics.     

Autophosphorylated residues involved in the regulation of human chk2 in vitro

Human checkpoint kinase 2 is a major actor in checkpoint activation through phosphorylation by ataxia telangiectasia mutated in response to DNA double-strand breaks. In the absence of de novo DNA damage, its autoactivation, reported in the event of increased Cds1/checkpoint kinase 2 (Chk2) expression, has been attributed to oligomerization. Here we report a study performed on autoactivated recombinant Chk2 proteins that aims to correlate kinase activity and phosphorylation status. Using a fluorescence-based technique to assay human checkpoint kinase 2 catalytic activity, slight differences in the ability to phosphorylate Cdc25C were observed, depending on the recombinant system used. Using mass spectrometry, the phosphorylation sites were mapped to identify sites potentially involved in the kinase activity. Five phosphorylated positions, at Ser120, Ser260, Thr225, Ser379 and Ser435, were found to be common to bacteria and insect cells expression systems. They were present in addition to the six known phosphorylation sites induced by ionizing radiation (Thr68, Thr432, Thr387, Ser516, Ser33/35 and Ser19) detected by immunoblotting. After phosphatase treatment, Chk2 regained activity via autorephosphorylation. The determination of the five common sites and ionizing-radiation-inducible positions as rephosphorylated confirms that they are potential positive regulators of Chk2 kinase activity. For Escherichia coli's most highly phosphorylated 6His-Chk2, 13 additional phosphorylation sites were assigned, including 7 novel sites on top of recently reported phosphorylation sites.     

Characterization of a Hank’s Type Serine/Threonine Kinase and Serine/Threonine Phosphoprotein Phosphatase in Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen that causes infections in eye, urinary tract, burn, and immunocompromised patients. We have cloned and characterized a serine/threonine (Ser/Thr) kinase and its cognate phosphoprotein phosphatase. By using oligonucleotides from the conserved regions of Ser/Thr kinases of mycobacteria, an 800-bp probe was used to screen P. aeruginosa PAO1 genomic library. A 20-kb cosmid clone was isolated, from which a 4.5-kb DNA with two open reading frames (ORFs) were subcloned. ORF1 was shown to encode Ser/Thr phosphatase (Stp1), which belongs to the PP2C family of phosphatases. Overlapping with the stp1 ORF, an ORF encoding Hank's type Ser/Thr kinase was identified. Both ORFs were cloned in pGEX-4T1 and expressed in Escherichia coli. The overexpressed proteins were purified by glutathione-Sepharose 4B affinity chromatography and were biochemically characterized. The Stk1 kinase is 39 kDa and undergoes autophosphorylation and can phosphorylate eukaryotic histone H1. A site-directed Stk1 (K86A) mutant was shown to be incapable of autophosphorylation. A two-dimensional phosphoamino acid analysis of Stk1 revealed strong phosphorylation at a threonine residue and weak phosphorylation at a serine residue. The Stp1 phosphatase is 27 kDa and is an Mn(2+)-, but not a Ca(2+)- or a Mg(2+)-, dependent Ser/Thr phosphatase. Its activity is inhibited by EDTA and NaF, but not by okadaic acid, and is similar to that of PP2C phosphatase.     

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.     

Calcineurin—immunosuppressor complexes

Crystal structures of the Ser/Thr phosphatase calcineurin (protein phosphatase 2B) have recently been solved by X-ray crystallography, both in the free-protein state, and complexed with the immunophilin/immunosuppressant FKBP12/FK506. Core elements of the calcineurin phosphatase have been found to be similar to the corresponding elements of Ser/Thr phosphatase 1 and purple acid phosphatase. The structures provide a basis for understanding calcineurin inhibition by a ternary complex of immunophilin and immunosuppressant proteins.     

Survey, analysis and genetic organization of genes encoding eukaryotic-like signaling proteins on a cyanobacterial genome.

Bacteria usually use two-component systems for signal transduction, while eukaryotic organisms employ Ser/Thr and Tyr kinases and phosphatases for the same purpose. Many prokaryotes turn out to harbor Ser/Thr and Tyr kinases, Ser/Thr and Tyr phosphatases, and their accessory components as well. The sequence determination of the genome of the cyanobacterium Synechocystis sp. strain PCC 6803 offers the possibility to survey the extent of such molecules in a prokaryotic organism. This cyanobacterium possesses seven Ser/Thr kinases, seven Ser/Thr and Tyr phosphatases, one protein kinase interacting protein, one protein kinase regulatory subunit and several WD40-repeat-containing proteins. The majority of the protein phosphatases presented in this study were previously reported as hypothetical proteins. We analyze here the structure and genetic organization of these ORFs in the hope of providing a guidance for their functional analysis. Unlike their eukaryotic counterparts, many of these genes are clustered on the chromosome, and this genetic organization offers the opportunity to study their possible interaction. In several cases, genes of two-component transducers are found within the same cluster as those encoding a Ser/Thr kinase or a Ser/Thr phosphatase; the implication for signal transduction mechanism will be discussed.     

<Emphasis Type="Italic">AtTBP2</Emphasis> and <Emphasis Type="Italic">AtTRP2</Emphasis> in <Emphasis Type="Italic">Arabidopsis</Emphasis> encode proteins that bind plant telomeric DNA and induce DNA bending in vitro

Telomeric DNA-binding proteins (TBPs) are crucial components that regulate the structure and function of eukaryotic telomeres and are evolutionarily conserved. We have identified two homologues of AtTBP1 (for Arabidopsis thaliana telomeric DNA binding protein 1), designated as AtTBP2 and AtTRP2, which encode proteins that specifically bind to the telomeric DNA of this plant. These proteins show extensive homology with other known plant TBPs. The isolated C-terminal segments of these proteins were capable of sequence-specific binding to duplex telomeric plant DNA in vitro. DNA bending assays using the Arabidopsis TBPs revealed that AtTBP1 and AtTBP2 have DNA-bending abilities comparable to that of the human homologue hTRF1, and higher than those of AtTRP1 and AtTRP2.     

Tightly bound to DNA proteins: possible universal substrates for intranuclear processes

Tightly bound to DNA proteins (TBPs) are a protein group that remains attached to DNA after its deproteinization by phenol, chloroform or salting-out. TBP are bound to DNA with covalent phosphotriester or non-covalent ion and hydrogen bonds. They appear to be a vast protein group involved in numerous intranuclear processes. The TBPs fraction co-purified with DNA deproteinized by mild procedures is extremely heterogeneous, tissue and species-specific. The protein fraction co-purified with DNA after harsh deproteinization procedures appears to be formed from few polypeptides common to different species and tissues. Interaction sites between DNA and TBPs depend on the physiological status of the cell. The binding sites of TBPs to DNA do not co-localize with the nuclear matrix attachment regions. We hypothesize that TBPs form a universal substrate for intranuclear processes.     

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.     

Acetylation and phosphorylation of high-mobility group A1 proteins in PC-3 human tumor cells

In this paper, we examined the posttranslational modifications (PTMs) of high-mobility group A1 (HMGA1) proteins in PC-3 human prostate cancer cells that are either treated or not treated with a histone deacetylase inhibitor, sodium butyrate. We found that, from a reversed-phase C4 column, the HMGA1a protein eluted in two different fractions with distinct forms of PTMs: Ser98, Ser101, and Ser102 were phosphorylated and Arg25 was methylated for both fractions; only the minor fraction, however, is hyperphosphorylated where Ser35, Thr52, and Thr77 were also phosphorylated. In addition, Lys14 was acetylated in the major but not the minor HMGA1a fraction isolated from the PC-3 cells that were not treated with butyrate. Likewise, HMGA1b, which is a splicing variant of HMGA1a, was acetylated on Lys14 and phosphorylated on the corresponding residues, i.e., Thr41, Thr66, Ser87, Ser90, and Ser91. The acetylation and phosphorylation of the HMGA1a and HMGA1b proteins may affect their interactions with other protein factors, which in turn may modulate the binding of HMGA1 proteins to DNA and regulate gene expression. In addition, the specifically posttranslationally modified HMGA1 proteins may serve as molecular biomarkers for cancer diagnosis and prognosis.     

Inhibition of phosphatidylinositol 4,5-bisphosphate-stimulated phospholipase D2 activity by Ser/Thr phosphorylation

Treatment of HeLa cells overexpressing PLD2 with the Ser/Thr-specific protein phosphatase inhibitor, okadaic acid, augmented spontaneous phosphorylation of PLD2 with concomitant inhibition of phosphatidylinositol 4,5-bisphosphate (PIP(2))-stimulated PLD2 activity. Dephosphorylation of the immunoprecipitated, spontaneously phosphorylated PLD2 in COS-7 cells by catalytic subunit of protein phosphatase 1gamma1 resulted in the stimulation of the PLD2 catalytic activity. These observations suggest that Ser/Thr phosphorylation regulates PLD2 activity.