Regulation of neuronal nitric-oxide synthase by calmodulin kinases.

Phosphorylation of neuronal nitric-oxide synthase (nNOS) by Ca2+/calmodulin (CaM)-dependent protein kinases (CaM kinases) including CaM kinase Ialpha (CaM-K Ialpha), CaM kinase IIalpha (CaM-K IIalpha), and CaM kinase IV (CaM-K IV), was studied. It was found that purified recombinant nNOS was phosphorylated by CaM-K Ialpha, CaM-K IIalpha, and CaM-K IV at Ser847 in vitro. Replacement of Ser847 with Ala (S847A) prevented phosphorylation by CaM kinases. Phosphorylated recombinant wild-type nNOS at Ser847 (approximately 0.5 mol of phosphate incorporation into nNOS) exhibited a 30% decrease of Vmax with little change of both the Km for L-arginine and Kact for CaM relative to unphosphorylated enzyme. The activity of mutant S847D was decreased to a level 50-60% as much as the wild-type enzyme. The decreased NOS enzyme activity of phosphorylated nNOS at Ser847 and mutant S847D was partially due to suppression of CaM binding, but not to impairment of dimer formation which is thought to be essential for enzyme activation. Inactive nNOS lacking CaM-binding ability was generated by mutation of Lys732-Lys-Leu to Asp732-Asp-Glu (Watanabe, Y., Hu, Y., and Hidaka, H. (1997) FEBS Lett. 403, 75-78). It was phosphorylated by CaM kinases, as was the wild-type enzyme, indicating that CaM-nNOS binding was not required for the phosphorylation reaction. We developed antibody NP847, which specifically recognize nNOS in its phosphorylated state at Ser847. Using the antibody NP847, we obtained evidence that nNOS is phosphorylated at Ser847 in rat brain. Thus, our results suggest that CaM kinase-induced phosphorylation of nNOS at Ser847 alters the activity control of this enzyme.     

Activation of actin-activated MgATPase activity of myosin II by phosphorylation with MAPK-activated protein kinase-1b (RSK-2)

Regulatory light chain of myosin II (MRLC) was identified as a novel substrate of p90 ribosomal S6 kinase (RSK)-2, a Ser/Thr protein kinase which is phosphorylated and activated by mitogen-activated protein kinase (MAPK) in vitro and in vivo. Phosphopeptide map of MRLC phosphorylated by RSK-2 was identical to that by myosin light chain kinase (MLCK). Phosphoserine was recovered by the phosphoamino acid analysis of MRLC phosphorylated by RSK-2. Further, phosphorylation using recombinant glutathione S-transferase (GST) fusion proteins of HeLa MRLC2 revealed that RSK-2 phosphorylated wild-type MRLC2 (GST-wtMRLC2) but not its mutants GST-MRLC2(S19A) or GST-MRLC2(T18AS19A) (alanine substituted for Ser19 or both Ser19 and Thr18). These results revealed that RSK-2 phosphorylates MRLC at Ser19 as did MLCK. Phosphorylation of myosin II by RSK-2 resulted in activation of actin-activated MgATPase activity of myosin II. Interestingly, RSK-2 activity to phosphorylate MRLC was suppressed by phosphorylation with MAPK. RSK-2 might be a mediator that regulates myosin II activity through the MAPK cascade.     

Protein kinases phosphorylating acidic ribosomal proteins from yeast cells

Phosphorylation of ribosomal acidic proteins ofSaccharomyces cerevisiae is an important mechanism regulating a number of active ribosomes. The key role in the regulatory mechanism is played by specific phosphoprotein kinases and phosphoprotein phosphatases. Three different cAMP-independent protein kinases phosphorylating acidic ribosomal proteins have been identified and characterized. The protein kinase 60S (PK60S), RAP kinase, and casein kinase type 2 (CK2). All three protein kinases phosphorylate serine residues which are localized in the C-terminal end of phosphoproteins. Synthetic peptides were used to determinate the amino acid sequence of phosphoacceptor site for PK60S. Peptide AAEESDDD derived from phosphoproteins YP1β/β′ and YP2α turned out to be the best substrate for PK60S. A number of halogenated benzimidazoles and 2-azabenzimidazoles were tested as inhibitors of the three protein kinases. 4,5,6,7-Tetrabromo-2-azabenzimidazole inhibits phosphorylation only of these polypeptides phosphorylated by protein kinase 60S, namely YP1β/β′ and YP2α, but not the other, YP1α and YP2β phosphorylated by protein kinases RAP and CK2. RAP kinase has been found in an active form in the soluble fraction ofS. cerevisiae. The enzyme uses ATP as a phosphate donor and is less sensitive to heparin than casein kinase 2. RAP kinase monophosphorylates the four acidic proteins. The ribosome-bound proteins are a better substrate for the enzyme. Multifunctional CK2 kinase phosphorylate all four acidic proteins. The kinase phosphorylates preferentially serine or threonine residues surrounded by cluster of acidic residues. The enzyme activity is stimulatedin vitro by the presence of polylysine and inhibited by heparin. Presented at theSymposium on Regulation of Translation of Genetic Information by Protein Phosphorylation, 21 st Congress of the Czechoslovak Society for Microbiology, Hradec Králové (Czech Republic), September 6–10, 1998.     

Phosphorylation of P0 Glycoprotein in Peripheral Nerve Myelin

The P0 protein in mammalian PNS myelin is known to undergo several posttranslational modifications, such as glycosylation, acylation, sulfation, and phosphorylation. Phosphorylation of purified P0 protein in vitro was studied comparatively using three enzymes, i.e., calcium/phospholipid-dependent protein kinase (protein kinase C), calcium/calmodulin-dependent protein kinase II (CaM kinase II), and the catalytic subunit of cyclic AMP-dependent protein kinase (A kinase). The phosphorylation of P0 protein by CaM kinase II was the greatest, followed by that by protein kinase C; phosphorylation by A kinase, however, was much lower. In order to identify phosphorylation sites, P0 protein was phosphorylated with [32P]ATP and each kinase and then digested with lysylendopeptidase. The resulting phosphopeptides were isolated by HPLC. Subsequent amino acid sequence analysis and comparison with the known sequence of P0 protein revealed that Ser181 and Ser204 were strongly phosphorylated by both protein kinase C and CaM kinase II. In addition, Ser214 was also phosphorylated by protein kinase C, but not by CaM kinase II. Because all of these sites are located in the cytoplasmic domain of P0 protein, phosphorylation may be important for maintenance of the major dense line of PNS myelin.     

Phosphorylation of the pro-apoptotic protein BAD on serine 155, a novel site, contributes to cell survival

Phosphorylation of BAD, a pro-apoptotic member of the Bcl-2 protein family, on either Ser112 or Ser136 is thought to be necessary and sufficient for growth factors to promote cell survival. Here we report that Ser155, a site phosphorylated by protein kinase A (PKA), also contributes to cell survival. Ser112 is thought to be the critical PKA target, but we found that BAD fusion proteins containing Ala at Ser112 (S112A) or Ser136 (S136A) or at both positions (S112/136A) were still heavily phosphorylated by PKA in an in vitro kinase assay. BAD became insensitive to phosphorylation by PKA only when both Ser112 and Ser136, or all three serines (S112/136/155) were mutated to alanine. In HEK293 cells, BAD fusion proteins mutated at Ser155 were refractory to phosphorylation induced by elevation of cyclic AMP(cAMP) levels. Phosphorylation of the S112/136A mutant was >90% inhibited by H89, a PKA inhibitor. The S155A mutant induced more apoptosis than the wild-type protein in serum-maintained CHO-K1 cells, and apoptosis induced by the S112/136A mutant was potentiated by serum withdrawal. These data suggest that Ser155 is a major site of phosphorylation by PKA and serum-induced kinases. Like Ser112 and Ser136, phosphorylation of Ser155 contributes to the cancellation of the pro-apoptotic function of BAD.     

Location and function of three sites phosphorylated on rat acetyl-CoA carboxylase by the AMP-activated protein kinase

1. We have sequenced two tryptic/chymotryptic peptides (TC3 and TC3a) containing a third site phosphorylated on rat acetyl-CoA carboxylase by the AMP-activated protein kinase. Comparison with the complete sequence of rat acetyl-CoA carboxylase predicted from the cDNA sequence [López-Casillas et al. (1988) Proc. Natl Acad. Sci. USA 85, 5784-5788] shows that this site corresponds to Ser1215. 2. Comparison of the cDNA sequence with previous amino acid sequence data identifies the other two sites for the AMP-activated protein kinase as Ser79 and Ser1200. A total of eight serine residues phosphorylated in vitro by six protein kinases can now be identified: six of these (Ser23, Ser25, Ser29, Ser77, Ser79 and Ser95) are clustered in the amino terminal region, while two (Ser1200 and Ser1215) are located in the central region. 3. Prior phosphorylation of Ser77 and Ser1200 by cyclic-AMP-dependent protein kinase prevents subsequent phosphorylation of Ser79 and Ser1200, but not Ser1215, by the AMP-activated protein kinase. Phosphorylation of Ser1215 under these conditions is not associated with a change in enzyme activity. 4. Limited trypsin treatment of native acetyl-CoA carboxylase selectively cleaves off the highly phosphorylated amino-terminal region containing Ser79. 5. Phosphorylation at Ser79 and Ser1200 by the AMP-activated protein kinase dramatically decreases Vmax and increases the A0.5 for citrate. Phosphorylation at Ser77 and Ser1200 by cyclic-AMP-dependent protein kinase causes more modest changes in the A0.5 for citrate and the Vmax. Dephosphorylation, or removal of the amino-terminal region containing Ser77/79 using trypsin, reverses all of these effects. 6. These results suggest that the effects of the AMP-activated protein kinase on acetyl-CoA carboxylase activity are mediated entirely by phosphorylation of Ser79, and not Ser1200 and Ser1215. The smaller effects of cyclic-AMP-dependent protein kinase are mediated by phosphorylation of Ser77.     

A novel mTOR-regulated phosphorylation site in elongation factor 2 kinase modulates the activity of the kinase and its binding to calmodulin

Eukaryotic elongation factor 2 (eEF2) kinase is an unusual calcium- and calmodulin-dependent protein kinase that is regulated by insulin through the rapamycin-sensitive mTOR pathway. Here we show that insulin decreases the ability of eEF2 kinase to bind calmodulin in a rapamycin-sensitive manner. We identify a novel phosphorylation site in eEF2 kinase (Ser78) that is located immediately next to its calmodulin-binding motif. Phosphorylation of this site is increased by insulin in a rapamycin-sensitive fashion. Regulation of the phosphorylation of Ser78 also requires amino acids and the protein kinase phosphoinositide-dependent kinase 1. Mutation of this site to alanine strongly attenuates the effects of insulin and rapamycin both on the binding of calmodulin to eEF2 kinase and on eEF2 kinase activity. Phosphorylation of Ser78 is thus likely to link insulin and mTOR signaling to the control of eEF2 phosphorylation and chain elongation. This site is not a target for known kinases in the mTOR pathway, e.g., the S6 kinases, implying that it is phosphorylated by a novel mTOR-linked protein kinase that serves to couple hormones and amino acids to the control of translation elongation. eEF2 kinase is thus a target for mTOR signaling independently of previously known downstream components of the pathway.     

Regulation of tyrosine hydroxylase by stress-activated protein kinases

Recombinant human tyrosine hydroxylase (hTH1) was found to be phosphorylated by mitogen and stress-activated protein kinase 1 (MSK1) at Ser40 and by p38 regulated/activated kinase (PRAK) on Ser19. Phosphorylation by MSK1 induced an increase in Vmax and a decrease in Km for 6-(R)-5,6,7,8-tetrahydrobiopterin (BH4), while these kinetic parameters were unaffected as a result of phosphorylation by PRAK. Phosphorylation of both Ser40 and Ser19 induced a high-affinity binding of 14-3-3 proteins, but only the interaction of 14-3-3 with Ser19 increased the hTH1 activity. The 14-3-3 proteins also inhibited the rate of dephosphorylation of Ser19 and Ser40 by 82 and 36%, respectively. The phosphorylation of hTH1 on Ser19 caused a threefold increase in the rate of phosphorylation of Ser40. These studies provide new insights into the possible roles of stress-activated protein kinases in the regulation of catecholamine biosynthesis.     

Regulation of Drosophila Ca2+/Calmodulin-Dependent Protein Kinase II by Autophosphorylation Analyzed by Site-Directed Mutagenesis

Abstract: In this study we demonstrate that Drosophila calcium/calmodulin-dependent protein kinase II (CaMKII) is capable of complex regulation by autophosphorylation of the three threonines within its regulatory domain. Specifically, we show that autophosphorylation of threonine-287 in Drosophila CaMKII is equivalent to phosphorylation of threonine-286 in rat α CaMKII both in its ability to confer calcium independence on the enzyme and in the mechanistic details of how it becomes phosphorylated. Autophosphorylation of this residue occurs only within the holoenzyme structure and requires calmodulin (CaM) to be bound to the substrate subunit. Phosphorylation of threonine-306 and threonine-307 in the CaM binding domain of the Drosophila kinase occurs only in the absence of CaM, and this phosphorylation is capable of inhibiting further CaM binding. Additionally, our findings suggest that phosphorylation of threonine-306 and threonine-307 does not mimic bound CaM to alleviate the requirement for CaM binding to the substrate subunit for intermolecular threonine-287 phosphorylation. These results demonstrate that the mechanism of regulatory autophosphorylation of this kinase predates the split between invertebrates and vertebrates.     

Regulation of p90RSK phosphorylation by SARS-CoV infection in Vero E6 cells

The 90 kDa ribosomal S6 kinases (p90RSKs) are a family of broadly expressed serine/threonine kinases with two kinase domains activated by extracellular signal-regulated protein kinase in response to many growth factors. Our recent study demonstrated that severe acute respiratory syndrome (SARS)-coronavirus (CoV) infection of monkey kidney Vero E6 cells induces phosphorylation and dephosphorylation of signaling pathways, resulting in apoptosis. In the present study, we investigated the phosphorylation status of p90RSK, which is a well-known substrate of these signaling pathways, in SARS-CoV-infected cells. Vero E6 mainly expressed p90RSK1 and showed weak expression of p90RSK2. In the absence of viral infection, Ser221 in the N-terminal kinase domain was phosphorylated constitutively, whereas both Thr573 in the C-terminal kinase domain and Ser380 between the two kinase domains were not phosphorylated in confluent cells. Ser380, which has been reported to be involved in autophosphorylation by activation of the C-terminal kinase domain, was phosphorylated in confluent SARS-CoV-infected cells, and this phosphorylation was inhibited by , which is an inhibitor of p38 mitogen-activated protein kinases (MAPK). Phosphorylation of Thr573 was not upregulated in SARS-CoV-infected cells. Thus, in virus-infected cells, phosphorylation of Thr573 was not necessary to induce phosphorylation of Ser380. On the other hand, Both Thr573 and Ser380 were phosphorylated by treatment with epidermal growth factor (EGF) in the absence of p38 MAPK activation. Ser220 was constitutively phosphorylated despite infection. These results indicated that phosphorylation status of p90RSK by SARS-CoV infection is different from that by stimulation of EGF. This is the first detailed report regarding regulation of p90RSK phosphorylation by virus infection.     

Mutational analysis of sites in sepiapterin reductase phosphorylated by Ca2+/calmodulin-dependent protein kinase II

Sepiapterin reductase (SPR) catalyzes the last step in the pathway of tetrahydrobiopterin biosynthesis in tissues. SPR is phosphorylated by Ca2+-dependent protein kinases, which indicates that Ca2+-activated protein kinases may play a role in the regulation of SPR in vivo. Phosphorylation sites of rat sepiapterin reductase (rSPR) by Ca2+/calmodulin-dependent protein kinase II were determined in the present study. Using specific monoclonal anti-phospho-Ser and -Thr antibodies, we found that only Ser residues of rSPR were phosphorylated. We constructed several point mutants of SPR by systematically replacing the three Ser residues by Ala ones. These mutants showed that all three Ser residues, i.e. S46, S196, and S214, of rSPR were phosphorylated. We also recognized that only Ser-213 of human SPR was phosphorylated. Each of these serine residues in SPR was found in the consensus sequence (Arg-X-X-Ser/Thr) of the phosphorylation site.     

Recent progress in characterization of protein kinase cascades for phosphorylation of ribosomal protein S6

Ribosomal protein S6 is phosphorylated in response to mitogens by activation of one or more protein kinase cascades. Phosphorylation of S6 in vivo is catalyzed by (at least) two distinct mitogen-activated S6 kinase families distinguishable by size, the 70 kDa and 90 kDa S6 kinases. Both S6 kinases are activated by serine/threonine phosphorylation. Members of each family have been cloned. The 90 kDa S6 kinases are activated more rapidly than the 80 kDa S6 kinase, and may have other intracellular targets. The 70 kDa S6 kinase is relatively specific for 40 S ribosomal subunits. No kinase capable of activating the 70 kDa S6 kinase has been identified. Members of the 90 kDa S6 kinases are activated in vitro by 42 kDa and 44 kDa MAP kinases, which are in turn activated by mitogen-dependent activators. The pathways for mitogen-stimulated S6 phosphorylation are discussed.     

Insulin-activated protein kinases phosphorylate a pseudosubstrate synthetic peptide inhibitor of the p70 S6 kinase

p70 S6 kinase, a major insulin-mitogen-activated ribosomal S6 protein kinase in mammalian cells, is activated by phosphorylation of multiple Ser/Thr residues on the enzyme polypeptide. A synthetic peptide, corresponding to a 37-residue segment from the carboxyl-terminal tail of the kinase which resembles the sequence phosphorylated in S6, acts as a competitive inhibitor of p70 S6 kinase without itself being phosphorylated by the enzyme. This synthetic peptide is phosphorylated by an array of protein kinases which are rapidly activated by insulin. Thus, these sequences of p70 S6 kinase constitute a potential autoinhibitory pseudosubstrate site, whose phosphorylation is catalyzed by candidate upstream-activating protein kinases.     

Ser289 phosphorylation activates both DAPK1 and DAPK2 but in response to different intracellular signaling pathways

DAPK1 and DAPK2 are calmodulin (CaM)-regulated protein kinases that share a high degree of homology in their catalytic and CaM regulatory domains. Both kinases function as tumor suppressors, and both have been implicated in autophagy regulation. Over the years, common regulatory mechanisms for the two kinases as well as kinase-specific ones have been identified. In a recent work, we revealed that DAPK2 is phosphorylated on Ser289 by the metabolic sensor AMPK, and that this phosphorylation enhances DAPK2 catalytic activity. Notably, Ser289 is conserved between DAPK1 and DAPK2, and was previously found to be phosphorylated in DAPK1 by RSK. Intriguingly, Ser289 phosphorylation was conversely reported to inhibit the pro-apoptotic activity of DAPK1 in cells. However, as the direct effect of this phosphorylation on DAPK1 catalytic activity was not tested, indirect effects were not excluded. Here, we compared Ser289 phosphorylation of the two kinases in the same cells and found that the intracellular signaling pathways that lead to Ser289 phosphorylation are mutually-exclusive and different for each kinase. In addition, we found that Ser289 phosphorylation in fact enhances DAPK1 catalytic activity, similar to the effect on DAPK2. Thus, Ser289 phosphorylation activates both DAPK1 and DAPK2, but in response to different intracellular signaling pathways.     

Dihydropyridine-sensitive calcium channels from skeletal muscle. II. Functional effects of differential phosphorylation of channel subunits.

Dihydropyridine-sensitive Ca2+ channels from skeletal muscle are multisubunit proteins and are regulated by protein phosphorylation. The purpose of this study was to determine: 1) which subunits are the preferential targets of various protein kinases when the channels are phosphorylated in vitro in their native membrane-bound state and 2) the consequences of these phosphorylations in functional assays. Using as substrates channels present in purified transverse (T) tubule membranes, cAMP-dependent protein kinase (PKA), protein kinase C (PKC), and a multifunctional Ca2+/calmodulin-dependent protein kinase (CaM protein kinase) preferentially phosphorylated the 165-kDa alpha 1 subunit to an extent that was 2-5-fold greater than the 52-kDa beta subunit. A protein kinase endogenous to the skeletal muscle membranes preferentially phosphorylated the beta peptide and showed little activity toward the alpha 1 subunit; however, the extent of phosphorylation was low. Reconstitution of partially purified channels into liposomes was used to determine the functional consequences of phosphorylation by these kinases. Phosphorylation of channels by PKA or PKC resulted in an activation of the channels that was observed as increases in both the rate and extent of Ca2+ influx. However, phosphorylation of channels by either the CaM protein kinase or the endogenous kinase in T-tubule membranes was without effect. Phosphorylation did not affect the sensitivities of the channels toward the dihydropyridines. Taken together, the results demonstrate that the alpha 1 subunit is the preferred substrate of PKA, PKC, and CaM protein kinase when the channels are phosphorylated in the membrane-bound state and that phosphorylation of the channels by PKA and PKC, but not by CaM protein kinase or an endogenous T-tubule membrane protein kinase, results in activation of the dihydropyridine-sensitive Ca2+ channels from skeletal muscle.     

Targeting inhibition of GluR1 Ser845 phosphorylation with an RNA aptamer that blocks AMPA receptor trafficking

Phosphorylation at glutamate receptor subunit 1(GluR1) Ser845 residue has been widely accepted to involve in GluR1-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor trafficking, but the in vivo evidence has not yet been established. One of the main obstacles is the lack of effective methodologies to selectively target phosphorylation at single amino acid residue. In this study, the Escherichia  coli -expressed glutathione- S -transferase-tagged intracellular carboxyl-terminal domain of GluR1 (cGluR1) was phosphorylated by protein kinase A for in vitro selection. We have successfully selected aptamers which effectively bind to phospho-Ser845 cGluR1 protein, but without binding to phospho-Ser831 cGluR1 protein. Moreover, pre-binding of the unphospho-cGluR1 protein with these aptamers inhibits protein kinase A-mediated phosphorylation at Ser845 residue. In contrast, the pre-binding of aptamer A2 has no effect on protein kinase C-mediated phosphorylation at Ser831 residue. Importantly, the representative aptamer A2 can effectively bind the mammalian GluR1 that inhibited GluR1/GluR1-containing AMPA receptor trafficking to the cell surface and abrogated forskolin-stimulated phosphorylation at GluR1 Ser845 in both green fluorescent protein–GluR1-transfected human embryonic kidney cells and cultured rat cortical neurons. The strategy to use aptamer to modify single-residue phosphorylation is expected to facilitate evaluation of the potential role of AMPA receptors in various forms of synaptic plasticity including that underlying psychostimulant abuse.     

The tumor suppressor DAP kinase is a target of RSK-mediated survival signaling

The viability of vertebrate cells depends on a complex signaling interplay between survival factors and cell-death effectors. Subtle changes in the equilibrium between these regulators can result in abnormal cell proliferation or cell death, leading to various pathological manifestations. Death-associated protein kinase (DAPK) is a multidomain calcium/calmodulin (CaM)-dependent Ser/Thr protein kinase with an important role in apoptosis regulation and tumor suppression. The molecular signaling mechanisms regulating this kinase, however, remain unclear. Here, we show that DAPK is phosphorylated upon activation of the Ras-extracellular signal-regulated kinase (ERK) pathway. This correlates with the suppression of the apoptotic activity of DAPK. We demonstrate that DAPK is a novel target of p90 ribosomal S6 kinases (RSK) 1 and 2, downstream effectors of ERK1/2. Using mass spectrometry, we identified Ser-289 as a novel phosphorylation site in DAPK, which is regulated by RSK. Mutation of Ser-289 to alanine results in a DAPK mutant with enhanced apoptotic activity, whereas the phosphomimetic mutation (Ser289Glu) attenuates its apoptotic activity. Our results suggest that RSK-mediated phosphorylation of DAPK is a unique mechanism for suppressing the proapoptotic function of this death kinase in healthy cells as well as Ras/Raf-transformed cells.     

Mitogen-independent phosphorylation of S6K1 and decreased ribosomal S6 phosphorylation in senescent human fibroblasts

The p70 ribosomal S6 kinase (S6K1) is rapidly activated following growth factor stimulation of quiescent fibroblasts and inhibition of this enzyme results in a G(1) arrest. Phosphorylation of the ribosomal S6 protein by S6K1 regulates the translation of both ribosomal proteins and initiation factors, leading to an increase in protein synthesis. We have examined the activation of S6K1 in human fibroblasts following mitogen stimulation. In early passage fibroblasts S6K1 is activated following serum stimulation as evidenced by increased kinase activity and site-specific phosphorylation. In contrast, site-specific phosphorylation of S6K1 at Thr421/Ser424 is diminished in senescent fibroblast cultures. A second phosphorylation site within S6K1 (Ser411) is phosphorylated even in the absence of serum stimulation and the enzyme shows increased phosphorylation as judged by decreased electrophoretic mobility. Inhibitor studies indicate that this phosphorylation is dependent upon the mammalian target of rapamycin, PI 3-kinase, and the MAPK pathway. In order to understand the consequences of the altered phosphorylation of the S6K1, we examined the phosphorylation state of the ribosomal S6 protein. In early passage fibroblasts the ribosomal S6 protein is phosphorylated upon serum stimulation while the phosphorylation of the ribosomal S6 protein is drastically reduced in senescent fibroblasts. These results suggest that the intracellular regulators of S6K1 are altered during replicative senescence leading to a deregulation of the enzyme and a loss of ribosomal S6 phosphorylation.     

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.     

Phosphorylation of serine 526 is required for MEKK3 activity, and association with 14-3-3 blocks dephosphorylation

MAPK/ERK kinase kinase 3 (MEKK3) is a mitogen-activated protein kinase kinase kinase (MAP3K) that functions upstream of the MAP kinases and IkappaB kinase. Phosphorylation is believed to be a critical component for MEKK3-dependent signal transduction, but little is known about the phosphorylation sites of this MAP3K. To address this question, point mutations were introduced in the activation loop (T-loop), substituting alanine for serine or threonine, and the mutants were transfected into HEK293 Epstein-Barr virus nuclear antigen cells. MEKK3-dependent activation of an NF-kappaB reporter gene as well as ERK, JNK, and p38 MAP kinases correlated with a requirement for serine at position 526. Constitutively active mutants of MEKK3, consisting of S526D and S526E, were capable of activating a NF-kappaB luciferase reporter gene as well as ERK and MEK, suggesting that a negative charge at Ser526 was necessary for MEKK3 activity and implicating Ser526 as a phosphorylation site. An antibody was developed that specifically recognized phospho-Ser526 of MEKK3 but did not recognize the S526A point mutant. The catalytically inactive (K391M) mutant of MEKK3 was not phosphorylated at Ser526, indicating that phosphorylation of Ser526 occurs via autophosphorylation. Endogenous MEKK3 was phosphorylated on Ser526 in response to osmotic stress. In addition, phosphorylation of Ser526 was required for MKK6 phosphorylation in vitro, whereas dephosphorylation of Ser526 was mediated by protein phosphatase 2A and sensitive to okadaic acid and sodium fluoride. Finally, the association between MEKK3 and 14-3-3 was dependent on Ser526 and prevented dephosphorylation of Ser526. In summary, Ser526 of MEKK3 is an autophosphorylation site within the T-loop that is regulated by PP2A and 14-3-3 proteins.