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PubMed1998

Immunohistochemical studies on phosphorylation of tyrosine hydroxylase in central catecholamine neurons using site- and phosphorylation state-specific antibodies.

Antibodies raised to phosphorylated forms of tyrosine hydroxylase, the first and rate-limiting enzyme in the catecholamine biosynthesis, were applied in immunohistochemical studies on rat brain slices incubated in vitro with a phosphodiesterase inhibitor (3-isobutyl-1-methylxanthine, IBMX) and on forskolin on formalin-perfused rat brains. Four antisera/antibodies were used: polyclonal rabbit antisera to (i) tyrosine hydroxylase phosphorylated at serine 40 (THS40p antiserum), (ii) tyrosine hydroxylase phosphorylated at serine 19 (THS19p antiserum), (iii) the native enzyme (pan-tyrosine hydroxylase antiserum), and mouse monoclonal antibody to (iv) native tyrosine hydroxylase. In the in vitro studies THS40p-like immunoreactivity was not observed unless slices were treated with IBMX-forskolin after which a dense fibre network was found in the striatum, and immunoreactive cell bodies were found in the ventral mesencephalon, especially in the ventral tegmental area. Although these cells were pan-tyrosine hydroxylase-positive, several of them were not stained with the tyrosine hydroxylase-monoclonal antibody. Moreover, there was a marked reduction of tyrosine hydroxylase-monoclonal antibody-immunoreactive fibres in drug-treated slices, suggesting that this tyrosine hydroxylase-monoclonal antibody does not recognize the serine 40-phosphorylated form of tyrosine hydroxylase. Treated slices did not show any THS40p-immunoreactive cell bodies in the dopaminergic A11 cell group and only a few, weakly fluorescent neurons were observed in locus coeruleus. However, a sparse fibre plexus was observed in locus coeruleus, possibly reflecting epinephrine fibres. In the perfused brains THS40p-like immunoreactivity could be visualized in some dopamine neurons in the ventral mesencephalon, especially the A10 area, and in noradrenergic locus coeruleus neurons, whereas THS19p-like immunoreactivity was found in all catecholamine groups studied, similar to the results obtained with the pan-tyrosine hydroxylase antiserum and the tyrosine hydroxylase-monoclonal antibody. In forebrain areas known to be innervated by mesencephalic dopamine neurons, no THS40p-positive fibres were observed, whereas THS19p-immunoreactive fibres were found in subregions of the striatum, olfactory tubercle and nucleus accumbens, essentially overlapping with dopamine fibres previously shown to contain cholecystokinin-like immunoreactivity. The present results suggests that antibodies directed against phosphorylated forms of tyrosine hydroxylase can be used to evaluate the state of tyrosine hydroxylase phosphorylation in individual neuronal cell bodies and processes both in vitro and in vivo.

1-Methyl-3-isobutylxanthine↗
PubMed2003

Ischemia-reperfusion decreases protein tyrosine phosphorylation and p38 mitogen-activated protein kinase phosphorylation in rat lung transplants.

BACKGROUND: Dramatic alterations of protein tyrosine phosphorylation have been found during the ischemia-reperfusion (IR) period of human lung transplantation. IR also induces activation of p38 mitogen-activated protein kinase (p38) in the heart and kidney. The objective of the present study was to determine whether these changes exist in a rat single-lung transplant model for further mechanistic investigations. METHODS: Isogeneic lung transplantation was performed from Lewis (LEW) to LEW rats, whereas allogeneic transplantation was from LEW to Brown Norway (BN) rats. Blood gases and peak airway pressure were monitored. Lung tissues were collected after 6 hours of cold ischemic preservation, after 30 minutes of warm ischemia for lung implantation, and after 2 hours of reperfusion. Protein tyrosine kinase (PTK) and phosphatase (PTP) activities were measured. Protein tyrosine phosphorylation, Src PTK and p38 expression and p38 phosphorylation were examined by western blotting. RESULTS: In both iso- and allografts, the lung function of transplants was very well preserved. Protein tyrosine phosphorylation, PTK and PTP activities were decreased significantly after 2 hours of reperfusion. Src protein level and phosphorylation of p38 were reduced after 2 hours of reperfusion. CONCLUSIONS: During the early IR period of lung transplantation, decreased protein tyrosine phosphorylation may be involved in apoptosis and other biologic changes. The lack of p38 activation suggests that activity of mitogen-activated protein kinase pathways in the lung transplantation setting may be different from other IR processes.

Animals↗
PubMed1992

Effect of phosphorylation on receptor conformation: the metarhodopsin I in equilibrium with metarhodopsin II equilibrium in multiply phosphorylated rhodopsin.

The superfamily of membrane-bound receptors, which function in signal transduction by activating a guanine nucleotide binding protein or G-protein in response to agonist binding, shares a number of structural and mechanistic properties. Among these similarities is downregulation of functional activity via receptor phosphorylation. In this study, the effects of intermediate levels of phosphorylation (greater than or equal to 4 added phosphates per receptor molecule) on receptor conformational equilibria are examined by comparing the photochemical properties of phosphorylated and unphosphorylated rhodopsins which were incorporated separately into 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine vesicles. Postflash spectra reflecting the contributions of metarhodopsins I, II, and III (meta I, meta II, and meta III) were obtained from these samples. Deconvolution of appropriate difference spectra allowed a determination of the concentration of the photointermediates of interest. Meta II is the form of photolyzed rhodopsin which binds and activates the visual G-protein (Gt); thus, its relative abundance at equilibrium and temporal stability are important parameters in determining the efficiency of visual signal transduction. The effects of pH and temperature on the meta I in equilibrium with meta II equilibrium constant (Keq) and the rate of decay of meta II to meta III were examined for the reconstituted phosphorylated and unphosphorylated rhodopsin samples. Keq was essentially unaffected by phosphorylation when measured at pH 7.0 and 8.0 and 20 and 37 degrees C. The decay time (lifetime) of meta II----meta III had a value of approximately 4.7 min in both phosphorylated and unphosphorylated samples.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

1,25-dihydroxyvitamin D3 modulates phosphorylation of serine 205 in the human vitamin D receptor: site-directed mutagenesis of this residue promotes alternative phosphorylation.

The vitamin D receptor (VDR) from a variety of animal species is a hormone-modulated substrate for phosphorylation in vivo. In this report, we utilize an expression vector to produce recombinant human VDR (hVDR) in 1,25-dihydroxyvitamin D3-treated COS-1 cells. Immunoprecipitation of the phosphorylated hVDR followed by gel purification and phosphoamino acid analysis revealed modification exclusively on one or more serine residues, consistent with previous studies of the VDR in other species. To identify the region of phosphorylation, immunoprecipitated and gel-purified hVDR from COS-1 cells was first mixed with purified hVDR isolated to homogeneity from Saccharomyces cerevisiae and then digested with trypsin or V8 protease, and the peptides were resolved on HPLC. The single phosphate-containing peptides were recovered and subjected to amino acid sequence analysis, revealing the modification to reside in a region extending from residue 171 to residue 206 common to both the tryptic- and the V8 protease-derived peptides. Sequential cleavage of similar VDR mixtures using trypsin and then CNBr, alpha-chymotrypsin, or thermolysin demonstrated an amino-terminal boundary of the phosphorylated peptide at 202. Selective manual Edman degradation of phosphorylated peptides beginning at 171, 195, and 200 revealed phosphate release only at serine 205. This peptide contained an average of 8-fold less radioactive phosphate in the absence of prior treatment of the culture cells with 1,25(OH)2D3. Site-directed modification of VDR serine 205 to alanine, aspartate, or glutamate each led to fully functional proteins when assessed in a transactivation assay using several VDRE-linked natural promoters. Unexpectedly, evaluation of the serine 205 to alanine hVDR mutant revealed that this protein continued to be phosphorylated in a hormone-dependent manner on an alternative site. These studies show directly that hVDR serine residue 205, a consensus site for casein kinase II, is modified in vivo in response to hormone.

Amino Acid Sequence↗
PubMed2004

In vitro phosphorylation of insulin receptor substrate 1 by protein kinase C-zeta: functional analysis and identification of novel phosphorylation sites.

Protein kinase C-zeta (PKC-zeta) participates both in downstream insulin signaling and in the negative feedback control of insulin action. Here we used an in vitro approach to identify PKC-zeta phosphorylation sites within insulin receptor substrate 1 (IRS-1) and to characterize the functional implications. A recombinant IRS-1 fragment (rIRS-1(449)(-)(664)) containing major tyrosine motifs for interaction with phosphatidylinositol (PI) 3-kinase strongly associated to the p85alpha subunit of PI 3-kinase after Tyr phosphorylation by the insulin receptor. Phosphorylation of rIRS-1(449)(-)(664) by PKC-zeta induced a prominent inhibition of this process with a mixture of classical PKC isoforms being less effective. Both PKC-zeta and the classical isoforms phosphorylated rIRS-1(449)(-)(664) on Ser(612). However, modification of this residue did not reduce the affinity of p85alpha binding to pTyr-containing peptides (amino acids 605-615 of rat IRS-1), as determined by surface plasmon resonance. rIRS-1(449)(-)(664) was then phosphorylated by PKC-zeta using [(32)P]ATP and subjected to tryptic phosphopeptide mapping based on two-dimensional HPLC coupled to mass spectrometry. Ser(498) and Ser(570) were identified as novel phosphoserine sites targeted by PKC-zeta. Both sites were additionally confirmed by phosphopeptide mapping of the corresponding Ser --> Ala mutants of rIRS-1(449)(-)(664). Ser(570) was specifically targeted by PKC-zeta, as shown by immunoblotting with a phosphospecific antiserum against Ser(570) of IRS-1. Binding of p85alpha to the S570A mutant was less susceptible to inhibition by PKC-zeta, when compared to the S612A mutant. In conclusion, our in vitro data demonstrate a strong inhibitory action of PKC-zeta at the level of IRS-1/PI 3-kinase interaction involving multiple serine phosphorylation sites. Whereas Ser(612) appears not to participate in the negative control of insulin signaling, Ser(570) may at least partly contribute to this process.

Amino Acid Sequence↗
PubMed1998

Two nonmuscle myosin II heavy chain isoforms expressed in rabbit brains: filament forming properties, the effects of phosphorylation by protein kinase C and casein kinase II, and location of the phosphorylation sites.

During the course of the expression of a 47-kDa COOH-terminal fragment of brain-type nonmuscle myosin heavy chain (MIIBF47), we found two closely related forms of MIIB, designated MIIB alpha and MIIB beta, in rabbit brains. The B alpha form corresponded to SMemb, described by Kuro-o et al. [(1991) J. Biol. Chem. 266, 3768] and was the more abundant form in rabbit brain, while the B beta form was novel. MIIB beta F47 differed from MIIB alpha F47 at six positions, three of which were within the carboxyl-terminal nonhelical domain; in MIIB beta F47, Ser, Pro, and Lys replaced Pro, Ser, and Glu, respectively. MIIB alpha F47 and MIIB beta F47 differed in filament assembly properties in the presence of various concentrations of salt, and a chimera containing the helical domain of MIIB beta F47 and the nonhelical domain of MIIB alpha F47 behaved very much like MIIB beta F47. Protein kinase C (PK C) incorporated 1 and 2 mol of phosphate/mol peptide of MIIB alpha F47 and MIIB beta F47, respectively, and caused similar levels of inhibition of assembly for both isoforms. Casein kinase II (CK II) incorporated 4 and 2 mol of phosphate/mol of MIIB alpha F47 and MIIB beta F47 peptides, respectively, and this caused strong inhibition of assembly for MIIB alpha F47 but only slight inhibition for MIIB beta F47. PK C sites in MIIB alpha F47 were localized within a region containing a cluster of Ser residues near the predicted junction of the helical and nonhelical domains: P-I-S(PO4)-F-S(PO4)-S(PO4)-S(PO4)-R-S(PO4)-. Out of the five potential PK C sites, only one site seemed to be phosphorylated per peptide. The PK C sites in MIIB beta F47 were localized as S(PO4)-I-S-F-S-S-(PO4)-R-S(PO4)-, with total incorporation of about 2 mol/mol of peptide. In addition, PK C phosphorylated a Ser within the predicted helical domain, E-V-S(PO4)-T-L, in both MIIB alpha F47 and MIIB beta F47. For CK II, five sites were identified within the COOH end of MIIB alpha F47: S(PO4)-L-E-L-S(PO4)-D-D-D-T(PO4)-E-S-K-T-S(PO4)-D-V-N-E-T-Q-P-P-Q-S(PO4) -E. The same sites were phosphorylated in MIIB beta F47 except for the first Ser, which was replaced by Pro in MIIB beta F47. An average of about two of the four potential sites were phosphorylated in MIIB beta F47, while in MIIB alpha F47 all five sites could be fully phosphorylated by CK II. Our results demonstrate that (1) the helical domains dictate the intrinsic salt dependence of assembly for nonmuscle myosin, (2) the isoforms are phosphorylatable by different kinases in an isoform specific manner mostly within the COOH-terminal nonhelical domain, and (3) the effects of the phosphorylation on assembly are isoform specific.

Amino Acid Sequence↗
PubMed2000

Phosphorylation of recombinant human ATP:citrate lyase by cAMP-dependent protein kinase abolishes homotropic allosteric regulation of the enzyme by citrate and increases the enzyme activity. Allosteric activation of ATP:citrate lyase by phosphorylated sugars.

Recombinantly expressed human ATP:citrate lyase was purified from E. coli, and its kinetic behavior was characterized before and after phosphorylation. Cyclic AMP-dependent protein kinase catalyzed the incorporation of only 1 mol of phosphate per mole of enzyme homotetramer, and glycogen synthase kinase-3 incorporated an additional 2 mol of phosphate into the phosphorylated protein. Isoelectric focusing revealed that all of the phosphates were incorporated into only one of the four enzyme subunits. Phosphorylation resulted in a 6-fold increase in V(max) and the conversion of citrate dependence from sigmoidal, displaying negative cooperativity, to hyperbolic. The phosphorylated recombinant enzyme is more similar to the enzyme isolated from mammalian tissues than unphosphorylated enzyme with respect to the K(m) for citrate, CoA, and ATP, and the specific activity. Fructose 6-phosphate was found to be a potent activator (60-fold) of the unphosphorylated recombinant enzyme, with half-maximal activation at 0.16 mM, which results in a decrease in the apparent K(m) for citrate and ATP, as well as an increase in the V(max) of the reaction. Thus, human ATP:citrate lyase activity is regulated in vitro allosterically by phosphorylated sugars as well as covalently by phosphorylation.

ATP Citrate (pro-S)-Lyase↗
PubMed1999

Differential phosphorylation of PKR associates with deregulation of eIF-2alpha phosphorylation and altered growth characteristics in 3T3-F442A fibroblasts.

Murine embryonic 3T3-F442A fibroblasts contain elevated levels of a factor (dRF) inhibitory to the phosphorylation of PKR, when cultured under differentiation restrictive (10% cat serum) as compared to permissive conditions (10% fetal bovine serum). Experiments were conducted with the objective of understanding the effect of altered PKR activity on the growth characteristics of 3T3-F442A fibroblasts. Analysis of the phosphoprotein pattern confirmed that the phosphorylation of PKR was reduced in cells cultured in cat serum during specific stages of growth. In a similar manner, evaluation of eIF-2alpha phosphorylation by vertical slab gel iso-electric focusing indicated that inactivation of PKR correlated with reduction of eIF-2alpha phosphorylation. The expression of PKR was confirmed by western blotting ruling out the possibility of diminished protein as the cause of loss of activity. In addition, the expression of dRF coincided with the inactivation of PKR as shown by immunoblotting and phosphorylation studies. The reduction in PKR activity and subsequent deregulation of eIF-2alpha phosphorylation was related to appearance of tumor-like cellular morphology and increased cell density as shown by cell counts and [3H]-thymidine uptake. Taken together, these results support a hypothesis that PKR functions to regulate the growth of 3T3-F442A cells. Furthermore, our findings raise the possibility that deregulation of PKR by endogenous inhibitory molecules, such as dRF, may alter normal growth and differentiation. Such a deregulation of PKR may also contribute to the proliferation of tumor cells.

3T3 Cells↗
PubMed1994

Protein kinase FA/glycogen synthase kinase-3 alpha after heparin potentiation phosphorylates tau on sites abnormally phosphorylated in Alzheimer's disease brain.

Previously, we identified protein kinase FA/glycogen synthase kinase-3 alpha (GSK-3 alpha) as a brain microtubule-associated tau kinase that phosphorylates Ser235 and Ser404 of tau and causes its electrophoretic mobility shift in gels, a unique property characteristic of paired helical filament-associated pathological tau (PHF-tau) in Alzheimer's disease brains. In this study, we found that the activity of kinase FA/GSK-3 alpha towards phosphorylation of brain tau could be stimulated approximately fourfold by heparin. The phosphorylation molar ratio was increased simultaneously up to 9 mol of phosphates/mol of tau, resulting in a reduced mobility of tau with an apparent molecular mass shift to approximately 68 kDa in sodium dodecyl sulfate gels, which is very similar to that observed in Alzheimer-tau. Tryptic digestion of 32P-labelled tau, followed by HPLC and two-dimensional separation on TLC cellulose plates, revealed eight major phosphopeptides. Phosphoamino acid analysis together with sequential manual Edman degradation and protein sequence analysis further revealed that, in addition to Ser235 and Ser404, heparin generated Thr212, Thr231, Ser262, Ser324, and Ser356, the five extra phosphorylation sites in tau. As Ser235, Ser262, Ser324, Ser356, and Ser404 (particularly the site of Ser262) have been identified as five of the most potent sites in tau responsible for reducing microtubule binding possibly involved in neuronal degeneration, and Thr231, Ser235, Ser262, and Ser404 are four of the most well documented sites abnormally phosphorylated in Alzheimer-tau, the results provide initial evidence that protein kinase FA/GSK-3 alpha after heparin potentiation may represent one of the most potent systems possibly involved in the abnormal phosphorylation of PHF-tau in Alzheimer's disease brains.

Alzheimer Disease↗
PubMed1995

Tyrosine hydroxylase phosphorylation in bovine adrenal chromaffin cells: the role of intracellular Ca2+ in the histamine H1 receptor-stimulated phosphorylation of Ser8, Ser19, Ser31, and Ser40.

Tyrosine hydroxylase (TOH), the rate-limiting enzyme in catecholamine biosynthesis, is regulated by phosphorylation. Activation of histaminergic H1 receptors on cultured bovine adrenal chromaffin cells stimulated a rapid increase in TOH phosphorylation (within 5 s) that was sustained for at least 5 min. The initial increase in TOH phosphorylation (up to 1 min) was essentially unchanged by the removal of extracellular Ca2+. In contrast, the H1-mediated response was abolished by preloading the cells with BAPTA acetoxymethyl ester (50 microM) and significantly reduced by prior exposure to caffeine (10 mM for 10 min) to deplete intracellular Ca2+. Tryptic-phosphopeptide analysis by HPLC revealed that the H1 response in the presence or absence of extracellular Ca2+ resulted in a major increase in the phosphorylation of Ser19 with smaller increases in that of Ser40 and Ser31. In contrast, although a brief stimulation with nicotine (30 microM for 60 s) also resulted in a major increase in Ser19 phosphorylation, this response was abolished in the absence of extracellular Ca2+. These data indicate that the mobilization of intracellular Ca2+ plays a crucial role in supporting H1-mediated TOH phosphorylation and may thus have a potentially important role in regulating catecholamine synthesis.

Adrenal Medulla↗
PubMed1998

Brain beta-spectrin phosphorylation: phosphate analysis and identification of threonine-347 as a heparin-sensitive protein kinase phosphorylation site.

Phosphorylation of brain spectrin was studied by a combination of in vivo and in vitro approaches. Chemical analysis of phosphate groups on electrophoretically purified mouse brain beta-spectrin yielded a stoichiometry of 3.2 +/- 0.18 mol of PO4/mol of beta-spectrin. The spectrin isolated by chromatographic methods from mouse brain, pig brain, and human erythrocytes yielded 4.1, 5.6, and 3.2 mol of PO4/mol of spectrin heterodimer, respectively. The 32P labeling of spectrin in retinal ganglion cell neurons or NB 2a/d1 neuroblastoma cells with [32P]orthophosphate showed phosphorylation of only beta-spectrin in vivo. Two-dimensional phosphopeptide map analyses showed that most of the in vivo sites on beta-spectrin were phosphorylated by either a heparin-sensitive endogenous cytoskeleton-associated protein kinase or protein kinase A. Phosphoamino acid analysis of in vivo and in vitro phosphorylated beta-spectrin showed that [32P]phosphate groups were incorporated into both serine (>90%) and threonine residues. In vitro, phosphate groups were incorporated into threonine residues by the heparin-sensitive endogenous protein kinase. The amino acid sequence VQQQLQAFNTY of an alpha-chymotryptic 32P-labeled peptide phosphorylated by the heparin-sensitive cytoskeleton-associated endogenous protein kinase corresponded to amino acid residues 338-348 on the beta1 repeat of beta-spectrinG (betaSPIIa) gene. These data suggest that phosphorylation of Thr347, which is localized on the presumptive synapsin I binding domain of beta-spectrinG, may play a role in synaptic function by regulating the binding of spectrin to synaptic vesicles.

Amino Acid Sequence↗
PubMed1996

Peroxynitrite disables the tyrosine phosphorylation regulatory mechanism: Lymphocyte-specific tyrosine kinase fails to phosphorylate nitrated cdc2(6-20)NH2 peptide.

To determine if nitration of tyrosine residues by peroxynitrite (PN), which can be generated endogenously, can disrupt the phosphorylation of tyrosine residues in proteins involved in cell signaling networks, we studied the effect of PN-promoted nitration of tyrosine residues in a pentadecameric peptide, cdc2(6-20)NH2, on the ability of the peptide to be phosphorylated. cdc2(6-20)NH2 corresponds to the tyrosine phosphorylation site of p34cdc2 kinase, which is phosphorylated by lck kinase (lymphocyte-specific tyrosine kinase, p56lck). PN nitrates both Tyr-15 and Tyr-19 of the peptide in phosphate buffer (pH 7.5) at 37 degrees C. Nitration of Tyr-15. which is the phosphorylated amino acid residue, inhibits completely the phosphorylation of the peptide. The nitration reaction is enhanced by either Fe(III)EDTA or Cu(II)-Zn(II)-superoxide dismutase (Cu,Zn-SOD). The kinetic data are consistent with the view that reactions of Fe(111)EDTA or Cu,Zn-SOD with the cis form of PN yield complexes in which PN decomposes more slowly to form N02+, the nitrating agent. Thus, the nitration efficiency of PN is enhanced. These results are discussed from the point of view that PN-promoted nitration will result in permanent impairment of cyclic cascades that control signal transduction processes and regulate cell cycles.

Amino Acid Sequence↗
PubMed1996

Phosphorylation of the respiratory burst oxidase subunit p47phox as determined by two-dimensional phosphopeptide mapping. Phosphorylation by protein kinase C, protein kinase A, and a mitogen-activated protein kinase.

The respiratory burst oxidase is responsible for superoxide (O2) production by phagocytes and B lymphocytes. This multicomponent enzyme is dormant in resting cells but is activated on exposure of the cells to an appropriate stimulus. Upon activation, several serine residues on the cytosolic oxidase subunit p47phox become phosphorylated. Using two-dimensional tryptic phosphopeptide mapping, we studied the phosphorylation of p47phox in 32Pi-loaded Epstein-Barr virus-transformed B lymphoblasts expressing wild type p47phox or any of several P47phox Ser -> Ala mutants. We were able to identify the labeled peptides from wild type p47phox as those contain- ing Ser303/304 Ser315, Ser320, Ser328 and/or Ser359/370, and Ser345/348 ; no 32P-labeled Ser310-containing peptide was found. When purified p47phox, was phosphorylated in vitro by various protein kinases, varying phosphopeptide patterns were observed. Protein kinase C phosphorylated all the peptides except the one containing Ser345/348; protein kinase A phosphorylated the peptide containing Ser320 and one or both of the peptides containing Ser328 and Ser359/370; while mitogen-activated protein kinase phophorylated only the peptide containing Ser345/348. These findings suggest that these three kinases play distinct roles in the activation of the respiratory burst oxidase, each of them catalyzing the phosphorylation of a different group of serines in p47phox.

Amino Acid Sequence↗
PubMed1998

Phosphorylation and inhibition of rat glucocorticoid receptor transcriptional activation by glycogen synthase kinase-3 (GSK-3). Species-specific differences between human and rat glucocorticoid receptor signaling as revealed through GSK-3 phosphorylation.

Transcriptional activation by the glucocorticoid receptor (GR) is regulated by both glucocorticoid binding and phosphorylation. The rat GR N-terminal transcriptional regulatory domain contains four major phosphorylation sites: threonine 171 (Thr171), serine 224 (Ser224), serine 232 (Ser232), and serine 246 (Ser246). We have previously demonstrated that Ser224 and Ser232 are phosphorylated by cyclin-dependent kinases, while Ser246 is phosphorylated by the c-Jun N-terminal kinase. We report here that the remaining GR phosphorylation site, Thr171, is a target for glycogen synthase kinase-3 (GSK-3) in vitro and in cultured mammalian cells. Increasing GSK-3 activity through its overexpression in cultured cells inhibits GR transcriptional enhancement, an effect dependent upon Thr171. Correspondingly, overexpression of a constitutively active form of the GSK-3 inhibitor, protein kinase B/Akt, increases GR transcriptional enhancement. Overexpression of GSK-3 had no effect on GR-mediated transcriptional repression of AP1-dependent gene expression. Importantly, transcriptional activation by the human GR (hGR), which contains an alanine (Ala150) at the position equivalent to Thr171 in rat GR, is not affected by GSK-3 overexpression. Introduction of a threonine residue at this position (A150T) establishes GSK-3-mediated inhibition of hGR transcriptional activation. These findings demonstrate species-specific differences in GR signaling, as revealed through GSK-3 phosphorylation, which suggests that GR function in rodents may not fully recapitulate receptor action in humans and that hGR is capable of adopting the GSK-3 signaling pathway through a somatic mutation.

Animals↗
PubMed1998

p40(phox) is phosphorylated on threonine 154 and serine 315 during activation of the phagocyte NADPH oxidase. Implication of a protein kinase c-type kinase in the phosphorylation process.

The superoxide-generating NADPH oxidase complex of phagocytic cells is a multicomponent system containing a membrane-bound flavocytochrome b and a small G protein Rac as well as cytosolic factors p67(phox) (phagocyte oxidase), p47(phox), and p40(phox), which translocate to the membrane upon activation. In a previous paper, we reported that p40(phox) undergoes phosphorylation on multiple sites upon stimulation of the NADPH oxidase by either phorbol 12-myristate 13-acetate or by formyl peptide with a time course that is strongly correlated with that of superoxide production (Fuchs, A., Bouin, A. P., Rabilloud, T., and Vignais, P. V. (1997) Eur. J. Biochem. 249, 531-539). In this study, through phosphoamino acid and tryptic peptide maps of in vivo and in vitro phosphorylated p40(phox), we show that p40(phox) is phosphorylated on serine and threonine residues during activation of the NADPH oxidase in dimethyl sulfoxide-differentiated HL60 promyelocytes as well as in isolated human neutrophils. In vitro phosphorylation studies using casein kinase II and protein kinase C (PKC) as well as the effect of various protein kinase inhibitors on the isoelectric focusing pattern of p40(phox) in whole cell lysates point to a role of a PKC type kinase in the phosphorylation of p40(phox). Directed mutagenesis of all PKC consensus sites enable us to conclude that Thr154 and Ser315 in p40(phox) are phosphorylated during activation of the NADPH oxidase.

Amino Acid Substitution↗
PubMed2000

Regulation of protein kinase D by multisite phosphorylation. Identification of phosphorylation sites by mass spectrometry and characterization by site-directed mutagenesis.

Activation of the serine/threonine kinase, protein kinase D (PKD/PKC mu) via a phorbol ester/PKC-dependent pathway involves phosphorylation events. The present study identifies five in vivo phosphorylation sites by mass spectrometry, and the role of four of them was investigated by site-directed mutagenesis. Four sites are autophosphorylation sites, the first of which (Ser(916)) is located in the C terminus; its phosphorylation modifies the conformation of the kinase and influences duration of kinase activation but is not required for phorbol ester-mediated activation of PKD. The second autophosphorylation site (Ser(203)) lies in that region of the regulatory domain, which in PKC mu interacts with 14-3-3tau. The last two autophosphorylation sites (Ser(744) and Ser(748)) are located in the activation loop but are only phosphorylated in the isolated PKD-catalytic domain and not in the full-length PKD; they may affect enzyme catalysis but are not involved in the activation of wild-type PKD by phorbol ester. We also present evidence for proteolytic activation of PKD. The fifth site (Ser(255)) is transphosphorylated downstream of a PKC-dependent pathway after in vivo stimulation with phorbol ester. In vivo phorbol ester stimulation of an S255E mutant no longer requires PKC-mediated events. In conclusion, our results show that PKD is a multisite phosphorylated enzyme and suggest that its phosphorylation may be an intricate process that regulates its biological functions in very distinct ways.

Alanine↗
PubMed2000

Phosphorylation of the pro-apoptotic protein BIK: mapping of phosphorylation sites and effect on apoptosis.

BIK is a pro-apoptotic BCL-2 family member and is the founding member of a subfamily of pro-apoptotic proteins known as "BH3-alone" proteins. Ectopic expression of BIK induces apoptosis in variety of mammalian cells. BIK complexes with various anti-apoptotic BCL-2 family proteins such as adenovirus E1B-19K and BCL-2 via the BH3 domain. However, the heterodimerization activity of BIK alone is insufficient for its apoptotic activity. Previous studies have shown that phosphorylation regulates the functional activity of both anti-apoptotic and pro-apoptotic members of the BCL-2 family. Here, we have examined phosphorylation of BIK and its effect on the apoptotic activity of BIK. We show that BIK exists as a phosphoprotein and is phosphorylated at residues 33 (threonine) and 35 (serine). Mutation of the phosphorylation sites, in which the Thr and Ser residues were changed to alanine residues, reduced the apoptotic activity of BIK without significantly affecting its ability to heterodimerize with BCL-2. Our results suggest that phosphorylation of BIK is required for eliciting efficient apoptotic activity. Partial purification of the protein kinase from HeLa cell cytoplasmic extracts suggest that BIK may be phosphorylated by a casein kinase II-related enzyme.

Apoptosis↗
PubMed2002

Phosphorylation of pyrimidine L-deoxynucleoside analog diphosphates. Kinetics of phosphorylation and dephosphorylation of nucleoside analog diphosphates and triphosphates by 3-phosphoglycerate kinase.

Anticancer and antiviral D- and L-nucleoside analogs are phosphorylated stepwise in the cells to the pharmacologically active triphosphate metabolites. We recently reported that in the last step, L-deoxynucleoside analog diphosphates are phosphorylated by 3-phosphoglycerate kinase (PGK). To explain the preference of PGK for L- over D-deoxynucleoside analog diphosphates, the kinetics of their phosphorylation were compared with the dephosphorylation of the respective triphosphates using recombinant human PGK. The results attributed favorable phosphorylation of L-deoxynucleoside analog diphosphates by PGK to differences in k(cat), which were consequences of varied orientations of the sugar and diphosphates in the catalytic site of PGK. The amino acids involved in the catalytic reaction of PGK (including Glu(344), Lys(220), and Asn(337)) were therefore mutated. The impact of mutations on the phosphorylation of L- and D-deoxynucleoside analog diphosphates was different from those on dephosphorylation of the respective triphosphates. This suggested that the interactions of the nucleoside analogs with amino acids during the transition state are different in the phosphorylation and dephosphorylation reactions. Thus, reversible action of the enzyme may not involve the same configuration of the active site. Furthermore, the amino acid determinants of the action of PGK for L-deoxynucleotides were not the same as for the D-deoxynucleotides. This study also suggests the potential impact of nucleoside analog diphosphates and triphosphates on the multiple cellular functions of PGK, which may contribute to the action of the analogs.

Amino Acid Substitution↗