Biomedical subjects

M Camps

Publications and source records attributed to M Camps.

At least 37 records · Page 2Linked to original sources

PubMed2000

Functional analysis of novel mutations in y(+)LAT-1 amino acid transporter gene causing lysinuric protein intolerance (LPI).

Lysinuric protein intolerance (LPI; MIM 222700) is an autosomal recessive disorder characterized by defective transport of the cationic amino acids lysine, arginine and ornithine at the basolateral membrane of the polar epithelial cells in the intestine and renal tubules, and by hyperammonemia after high-protein meals. LPI is caused by mutations in the SLC7A7 (solute carrier family 7, member 7) gene encoding y(+)LAT-1 (y(+)L amino acid transporter-1), which co-induces together with 4F2 heavy chain (4F2hc) system y(+)L in Xenopus oocytes. All Finnish LPI patients share the same founder mutation 1181-2A-->T (LPI(Fin)) not found in LPI patients elsewhere. Mutation screening of 20 non-Finnish LPI patients revealed 10 novel mutations: four deletions, two missense mutations, two nonsense mutations, a splice site mutation and a tandem duplication. Five LPI mutations (L334R, G54V, 1291delCTTT, 1548delC and LPI(Fin)) were studied functionally. All mutant proteins failed to co-induce amino acid transport activity when expressed with 4F2hc in Xenopus oocytes. Immunostaining experiments revealed that frameshift mutants 1291delCTTT, 1548delC and LPI(Fin)remained intracellular on expression with 4F2hc. In contrast, the missense mutants L334R and G54V reached the oocyte plasma membrane when co-expressed with 4F2hc, demonstrating that they are transport-inactivating mutations. This finding, together with the strong degree of conservation among all members of this family of amino acid transporters, indicates that residues L334 and G54 play a crucial role in the function of the y(+)LAT-1 transporter.

Amino Acid Metabolism, Inborn Errors↗

PubMed2000

Anaerobic degradation of fluorinated aromatic compounds.

Anaerobic enrichment cultures with sediment from an intertidal strait as inoculum were established under denitrifying, sulfate-reducing, iron-reducing and methanogenic conditions to examine the biodegradation of mono-fluorophenol and mono-fluorobenzoate isomers. Both phenol and benzoate were utilized within 2-6 weeks under all electron-accepting conditions. However, no degradation of the fluorophenols was observed within 1 year under any of the anaerobic conditions tested. Under denitrifying conditions, 2-fluorobenzoate and 4-fluorobenzoate were depleted within 84 days and 28 days, respectively. No loss of 3-fluorobenzoate was observed. All three fluorobenzoate isomers were recalcitrant under sulfate-reducing, iron-reducing, and methanogenic conditions. The degradation of the fluorobenzoate isomers under denitrifying conditions was examined in more detail using soils and sediments from different geographic regions around the world. Stable enrichment cultures were obtained on 2-fluorobenzoate or 4-fluorobenzoate with inoculum from most sites. Fluoride was released stoichiometrically, and nitrate reduction corresponded to the values predicted for oxidation of fluorobenzoate to CO2 coupled to denitrification. The 2-fluorobenzoate-utilizing and 4-fluorobenzoate-utilizing cultures were specific for fluorobenzoates and did not utilize other halogenated (chloro-, bromo-, iodo-) benzoic acids. Two denitrifying strains were isolated that utilized 2-fluorobenzoate and 4-fluorobenzoate as growth substrates. Preliminary characterization indicated that the strains were closely related to Pseudomonas stutzeri.

Anaerobiosis↗

PubMed2000

Dual specificity phosphatases: a gene family for control of MAP kinase function.

Mitogen-activated protein (MAP) kinases are important players in signal transduction pathways activated by a range of stimuli and mediate a number of physiological and pathological changes in cell function. MAP kinase activation requires phosphorylation on a threonine and tyrosine residue located within the activation loop of kinase subdomain VIII. This process is reversible even in the continued presence of activating stimuli, indicating that protein phosphatases provide an important mechanism for MAP kinase control. Dual specificity phosphatases (DSPs) are an emerging subclass of the protein tyrosine phosphatase (PTP) gene superfamily, which appears to be selective for dephosphorylating the critical phosphothreonine and phosphotyrosine residues within MAP kinases. Some DSPs are localized to different subcellular compartments and moreover, certain family members appear highly selective for inactivating distinct MAP kinase isoforms. This enzymatic specificity is due in part to powerful catalytic activation of the DSP phosphatase after tight binding of its amino-terminal to the target MAP kinase. DSP gene expression is induced strongly by various growth factors and/or cellular stresses, providing a sophisticated transcriptional mechanism for targeted inactivation of selected MAP kinase activities.

Amino Acid Sequence↗

PubMed1999

Identification of a membrane protein, LAT-2, that Co-expresses with 4F2 heavy chain, an L-type amino acid transport activity with broad specificity for small and large zwitterionic amino acids.

We have identified a new human cDNA, L-amino acid transporter-2 (LAT-2), that induces a system L transport activity with 4F2hc (the heavy chain of the surface antigen 4F2, also named CD98) in oocytes. Human LAT-2 is the fourth member of the family of amino acid transporters that are subunits of 4F2hc. The amino acid transport activity induced by the co-expression of 4F2hc and LAT-2 was sodium-independent and showed broad specificity for small and large zwitterionic amino acids, as well as bulky analogs (e.g. BCH (2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid)). This transport activity was highly trans-stimulated, suggesting an exchanger mechanism of transport. Expression of tagged N-myc-LAT-2 alone in oocytes did not induce amino acid transport, and the protein had an intracellular location. Co-expression of N-myc-LAT-2 and 4F2hc gave amino acid transport induction and expression of N-myc-LAT-2 at the plasma membrane of the oocytes. These data suggest that LAT-2 is an additional member of the family of 4F2 light chain subunits, which associates with 4F2hc to express a system L transport activity with broad specificity for zwitterionic amino acids. Human LAT-2 mRNA is expressed in kidney >>> placenta >> brain, liver > spleen, skeletal muscle, heart, small intestine, and lung. Human LAT-2 gene localizes at chromosome 14q11.2-13 (13 cR or approximately 286 kb from marker D14S1349). The high expression of LAT-2 mRNA in epithelial cells of proximal tubules, the basolateral location of 4F2hc in these cells, and the amino acid transport activity of LAT-2 suggest that this transporter contributes to the renal reabsorption of neutral amino acids in the basolateral domain of epithelial proximal tubule cells.

Amino Acid Sequence↗

PubMed1999

Non-type I cystinuria caused by mutations in SLC7A9, encoding a subunit (bo,+AT) of rBAT.

Cystinuria (MIM 220100) is a common recessive disorder of renal reabsorption of cystine and dibasic amino acids. Mutations in SLC3A1, encoding rBAT, cause cystinuria type I (ref. 1), but not other types of cystinuria (ref. 2). A gene whose mutation causes non-type I cystinuria has been mapped by linkage analysis to 19q12-13.1 (Refs 3,4). We have identified a new transcript, encoding a protein (bo, +AT, for bo,+ amino acid transporter) belonging to a family of light subunits of amino acid transporters, expressed in kidney, liver, small intestine and placenta, and localized its gene (SLC7A9) to the non-type I cystinuria 19q locus. Co-transfection of bo,+AT and rBAT brings the latter to the plasma membrane, and results in the uptake of L-arginine in COS cells. We have found SLC7A9 mutations in Libyan-Jews, North American, Italian and Spanish non-type I cystinuria patients. The Libyan Jewish patients are homozygous for a founder missense mutation (V170M) that abolishes b o,+AT amino-acid uptake activity when co-transfected with rBAT in COS cells. We identified four missense mutations (G105R, A182T, G195R and G295R) and two frameshift (520insT and 596delTG) mutations in other patients. Our data establish that mutations in SLC7A9 cause non-type I cystinuria, and suggest that bo,+AT is the light subunit of rBAT.

Amino Acid Sequence↗

PubMed1998

Regulated expression of dual specificity protein phosphatases in rat brain.

Activated mitogen-activated protein (MAP) kinases play an essential role controlling many neuronal functions. Dual specificity protein phosphatases (DS-PTPs) elicit selective inactivation of MAP kinases and are under tight transcriptional control. We have studied expression of four DS-PTPs (MKP-1, MKP-X, MKP-3 and B23) in rat brain and examined changes during post-natal development and following kainic acid induced seizure activity. In normal adult brain these DS-PTPs exhibit a strikingly different expression pattern. Only MKP-1 was regulated during development with levels increased transiently (P15-P21) within the thalamus and somatosensory cortex. Following kainate treatment, MKP-1, MKP-3 and B23 all exhibit striking changes in expression within hippocampal subfields CA1-3 and dentate gyrus. Regulated transcription of DS-PTPs may play a critical role controlling MAP kinase dependent processes including synaptic remodeling and neuronal death.

Aging↗

PubMed1998

Catalytic activation of the phosphatase MKP-3 by ERK2 mitogen-activated protein kinase.

MAP kinase phosphatase-3 (MKP-3) dephosphorylates phosphotyrosine and phosphothreonine and inactivates selectively ERK family mitogen-activated protein (MAP) kinases. MKP-3 was activated by direct binding to purified ERK2. Activation was independent of protein kinase activity and required binding of ERK2 to the noncatalytic amino-terminus of MKP-3. Neither the gain-of-function Sevenmaker ERK2 mutant D319N nor c-Jun amino-terminal kinase-stress-activated protein kinase (JNK/SAPK) or p38 MAP kinases bound MKP-3 or caused its catalytic activation. These kinases were also resistant to enzymatic inactivation by MKP-3. Another homologous but nonselective phosphatase, MKP-4, bound and was activated by ERK2, JNK/SAPK, and p38 MAP kinases. Catalytic activation of MAP kinase phosphatases through substrate binding may regulate MAP kinase activation by a large number of receptor systems.

Amino Acid Sequence↗

PubMed1998

The mitogen-activated protein kinase phosphatase-3 N-terminal noncatalytic region is responsible for tight substrate binding and enzymatic specificity.

We have reported recently that the dual specificity mitogen-activated protein kinase phosphatase-3 (MKP-3) elicits highly selective inactivation of the extracellular signal-regulated kinase (ERK) class of mitogen-activated protein (MAP) kinases (Muda, M., Theodosiou, A., Rodrigues, N., Boschert, U., Camps, M., Gillieron, C., Davies, K., Ashworth, A., and Arkinstall, S. (1996) J. Biol. Chem. 271, 27205-27208). We now show that MKP-3 enzymatic specificity is paralleled by tight binding to both ERK1 and ERK2 while, in contrast, little or no interaction with either c-Jun N-terminal kinase/stress activated protein kinase (JNK/SAPK) or p38 MAP kinases was detected. Further study revealed that the N-terminal noncatalytic domain of MKP-3 (MKP-3DeltaC) binds both ERK1 and ERK2, while the C-terminal MKP-3 catalytic core (MKP-3DeltaN) fails to precipitate either of these MAP kinases. A chimera consisting of the N-terminal half of MKP-3 with the C-terminal catalytic core of M3-6 also bound tightly to ERK1 but not to JNK3/SAPKbeta. Consistent with a role for N-terminal binding in determining MKP-3 specificity, at least 10-fold higher concentrations of purified MKP-3DeltaN than full-length MKP-3 is required to inhibit ERK2 activity. In contrast, both MKP-3DeltaN and full-length MKP-3 inactivate JNK/SAPK and p38 MAP kinases at similarly high concentrations. Also, a chimera of the M3-6 N terminus with the MKP-3 catalytic core which fails to bind ERK elicits non selective inactivation of ERK1 and JNK3/SAPKbeta. Together, these observations suggest that the physiological specificity of MKP-3 for inactivation of ERK family MAP kinases reflects tight substrate binding by its N-terminal domain.

Amino Acid Sequence↗

PubMed1998

Role of semicarbazide-sensitive amine oxidase on glucose transport and GLUT4 recruitment to the cell surface in adipose cells.

The previous characterization of an abundant population of non-adrenergic imidazoline-I2 binding sites in adipocytes and the recent demonstration of the interplay between these binding sites and amine oxidases led us to analyze the amine oxidase activity in membranes from isolated rat adipocytes. Adipocyte membranes had substantial levels of semicarbazide-sensitive amine oxidase (SSAO). SSAO activity and immunoreactive SSAO protein were maximal in plasma membranes, and they were also detectable in intracellular membranes. Vesicle immunoisolation analysis indicated that GLUT4-containing vesicles from rat adipocytes contain substantial levels of SSAO activity and immunoreactive SSAO protein. Immunotitration of intracellular GLUT4 vesicles indicated that GLUT4 and SSAO colocalize in an endosomal compartment in rat adipocytes. SSAO activity was also found in GLUT4 vesicles from 3T3-L1 adipocytes and rat skeletal muscle. Benzylamine, a substrate of SSAO activity, caused a marked stimulation of glucose transport in isolated rat adipocytes in the presence of very low vanadate concentrations that by themselves were ineffective in exerting insulin-like effects. This synergistic effect of benzylamine and vanadate on glucose transport was totally abolished in the presence of semicarbazide, a specific inhibitor of SSAO. Subcellular membrane fractionation revealed that the combination of benzylamine and vanadate caused a recruitment of GLUT4 to the plasma membrane of adipose cells. The stimulatory effects of benzylamine and vanadate on glucose transport were blocked by catalase, suggesting that hydrogen peroxide production coupled to SSAO activity plays a crucial regulatory role. Based on these results we propose that SSAO activity might contribute through hydrogen peroxide production to the in vivo regulation of GLUT4 trafficking in adipose cells.

Adipocytes↗

PubMed1998

Induction of the mitogen-activated protein kinase phosphatase MKP3 by nerve growth factor in differentiating PC12.

In PC12 sympathetic neurons activation and nuclear translocation of ERK family MAP kinases plays an essential role in processes underlying nerve growth factor (NGF)-dependent differentiation. We have recently cloned MKP-3 as a novel dual specificity phosphatase displaying selectivity towards inactivation of the ERK1 and ERK2 MAP kinases. Here we report that in PC12 cells, MKP-3 undergoes powerful and specific up-regulation by NGF while a number of mitogens and cellular stresses are ineffective. NGF-stimulated MKP-3 expression appears after 1 h, is maximal at 3 h, and is sustained for 5 days. This coincides with a critical period of neurite outgrowth and terminal differentiation. Consistent with a role mediating inhibition of PC12 cell MAP kinases, NGF-stimulated ERK2 activation was suppressed considerably following pretreatment with fibroblast growth factor and 9-cis-retinal, two additional differentiation factors found to induce powerfully MKP-3 expression. Given the clear cytosolic localization of MKP3 in PC12 cells and sympathetic neurons, these results suggest a critical role for inactivating ERK MAP kinases in non-nuclear compartments during essential stages of NGF-mediated PC12 differentiation.

Animals↗

PubMed1998

Molecular cloning and characterization of two phosphatase 2A catalytic subunit genes from Arabidopsis thaliana.

The plant Arabidopsis thaliana contains five isoforms of the catalytic subunit of protein phosphatase 2A (PP2A) that can be grouped into two families, one composed by isoforms PP2A-1, -2 and -5 and the other composed by isoforms PP2A-3 and PP2A-4. An Arabidopsis genomic library was screened and several clones corresponding to genes PP2A-3 and PP2A-4 were isolated and analysed. Both genes span over approximately 4.5kbp and are composed of 11 exons and 10 introns that show identical organization. Their untranslated regions are also highly conserved, suggesting that the two genes derive from a common ancestral gene. However, the position of intron/exon junctions completely differs from that of the human PP2A genes. Two transcription start sites have been found in the PP2A-3 gene, the major one mapping at nucleotide position -188 from the translation start codon, whereas only one is observed in PP2A-4 (-145). Functional gene promoter analysis reveals that elements required for transient expression of PP2A-3 and PP2A-4 on a protoplast system are contained within a region of about 600bp upstream from the transcription start sites. This is the first report on the cloning and characterization of genes encoding catalytic subunits of Ser/Thr protein phosphatases 2A in higher plants.

Amino Acid Sequence↗

PubMed1998

Specific T helper cell requirement for optimal induction of cytotoxic T lymphocytes against major histocompatibility complex class II negative tumors.

This study shows that induction of tumor-specific CD4+ T cells by vaccination with a specific viral T helper epitope, contained within a synthetic peptide, results in protective immunity against major histocompatibility complex (MHC) class II negative, virus-induced tumor cells. Protection was also induced against sarcoma induction by acutely transforming retrovirus. In contrast, no protective immunity was induced by vaccination with an unrelated T helper epitope. By cytokine pattern analysis, the induced CD4+ T cells were of the T helper cell 1 type. The peptide-specific CD4+ T cells did not directly recognize the tumor cells, indicating involvement of cross-priming by tumor-associated antigen-presenting cells. The main effector cells responsible for tumor eradication were identified as CD8+ cytotoxic T cells that were found to recognize a recently described immunodominant viral gag-encoded cytotoxic T lymphocyte (CTL) epitope, which is unrelated to the viral env-encoded T helper peptide sequence. Simultaneous vaccination with the tumor-specific T helper and CTL epitopes resulted in strong synergistic protection. These results indicate the crucial role of T helper cells for optimal induction of protective immunity against MHC class II negative tumor cells. Protection is dependent on tumor-specific CTLs in this model system and requires cross-priming of tumor antigens by specialized antigen-presenting cells. Thus, tumor-specific T helper epitopes have to be included in the design of epitope-based vaccines.

Amino Acid Sequence↗

PubMed1998

Activation of a phospholipase Cbeta2 deletion mutant by limited proteolysis.

All phosphoinositide-specific phospholipases C (PLC) identified until today exhibit a high degree of similarity within two regions of 170 and 260 residues, respectively, which are designated regions X and Y. The PLCbeta family, including four members designated PLCbeta1, PLCbeta2, PLCbeta3 and PLCbeta4, is regulated by heterotrimeric G proteins. In order to investigate structure-function relationships of PLCbeta2, we expressed PLCbeta2Delta, a deletion mutant of PLCbeta2 which lacks most of the sequence downstream of region Y, in the baculovirus/insect cell system. The mutant was present in both soluble and particulate fractions of Sf9 cells and was demonstrated to be catalytically active and sensitive to betagamma-subunits. Sf9 cytosol containing this mutant was subjected to limited proteolysis by trypsin and S. aureus protease V8, respectively. Immunochemical analysis revealed that both proteases cleaved the enzyme between the regions X and Y. Most interestingly, proteolytic cleavage at this site by both proteases stimulated the catalytic activity of PLC2beta2Delta. The proteolytically activated enzyme was still sensitive to betagamma-subunits and showed an unchanged concentration dependence on Ca2+. Gel filtration chromatography indicated that the fragments generated by cleavage between the regions X and Y were still connected and formed a functional heterodimeric complex. In order to visualize all fragments generated by protease V8, PLCbeta2Delta was purified to homogeneity from Sf9 cytosol. Limited proteolysis of the purified enzyme by S. aureus protease V8 and subsequent SDS/PAGE and silver staining revealed that several cuts take place between the regions X and Y and that the resulting fragments remain intact. We hypothesize that the activating proteolytic cut induces a conformational change of the enzyme which might facilitate hydrolysis of the phospholipid substrate.

Animals↗

PubMed1998

Searching for ways to upregulate GLUT4 glucose transporter expression in muscle.

1. Skeletal muscle is a major glucose-utilizing tissue in the absorptive state and alterations in muscle insulin-stimulated glucose uptake lead to derangements in whole body glucose disposal. 2. Furthermore, muscle GLUT4 overexpression in transgenic animals ameliorates insulin resistance associated with obesity or diabetes, which suggests that increasing GLUT4 in muscle by pharmacological intervention may be an effective therapy in insulin-resistant states. 3. This highlights the importance of understanding the pathways that upregulate GLUT4 glucose transporter expression in muscle. 4. We review studies describing the regulation of GLUT4 and the information currently available on the mechanisms that control GLUT4 expression in muscle.

Animals↗

PubMed1998

The amino acid transport system y+L/4F2hc is a heteromultimeric complex.

4F2hc is an almost ubiquitous transmembrane protein in mammalian cells; upon expression in Xenopus laevis oocytes, it induces amino acid transport with characteristics of system y+L. Indirect evidence fostered speculation that function requires the association of 4F2hc with another protein endogenous to oocytes and native tissues. We show that expression of system y+L-like amino acid transport activity by 4F2hc in oocytes is limited by an endogenous factor and that direct covalent modification of external cysteine residue(s) of an oocyte membrane protein blocks system y+L/4F2hc transport activity, based on the following. 1) Induction of system y+L-like activity saturates at very low doses of human 4F2hc cRNA (0.1 ng/oocyte). This saturation occurs with very low expression of 4F2hc at the oocyte surface, and further increased expression of the protein at the cell surface does not result in higher induction of system y+L-like activity. 2) Human 4F2hc contains only two cysteine residues (C109 and C330). We mutated these residues, singly and in combination, to serine (C109S; CS1, C330S; CS2 and C109S-C330S, Cys-less). Mutation CS2 had no effect on the expressed system y+L-like transport activity, whereas C109S-containing mutants (CS1 and Cys-less) retained only partial y+L-like transport activity (30 to 50% of wild type). 3) Hg2+, the organic mercury compounds pCMB, and the membrane-impermeant pCMBS almost completely inactivated system y+L-like induced by human 4F2hc wild type and all the mutants studied. This was reversed by ss-mercaptoethanol, indicating that external cysteine residue(s) are the target of this inactivation. 4) Sensitivity to Hg2+ inactivation is increased by pretreatment of oocytes with ss-mercaptoethanol or in the C109S-containing mutants (CS1 and Cys-less). The increased Hg2+ reactivity of C109S-containing mutants supports the possibility that C109 may be linked by a disulfide bond to the Hg2+-targeted cysteine residue of the associated protein. These results indicate that 4F2hc is intimately associated with a membrane oocyte protein for the expression of system y+L amino acid transport activity. To our knowledge, this is the first direct evidence for a heteromultimeric protein structure of an organic solute carrier in mammals.

4-Chloromercuribenzenesulfonate↗

PubMed1998

Overexpression of glycogen phosphorylase increases GLUT4 expression and glucose transport in cultured skeletal human muscle.

Skeletal muscle glucose utilization, a major factor in the control of whole-body glucose tolerance, is modulated in accordance with the muscle metabolic demand. For instance, it is increased in chronic contraction or exercise training in association with elevated expression of GLUT4 and hexokinase II (HK-II). In this work, the contribution of increased metabolic flux to the regulation of the glucose transport capacity was analyzed in cultured human skeletal muscle engineered to overexpress glycogen phosphorylase (GP). Myocytes treated with an adenovirus-bearing muscle GP cDNA (AdCMV-MGP) expressed 10 times higher GP activity and exhibited a twofold increase in the Vmax for 2-deoxy-D-[3H]glucose (2-DG) uptake, with no effect on the apparent Km. The stimulatory effect of insulin on 2-DG uptake was also markedly enhanced in AdCMV-MGP-treated cells, which showed maximal insulin stimulation 2.8 times higher than control cells. No changes in HKII total activity or the intracellular compartmentalization were found. GLUT4, protein, and mRNA were raised in AdCMV-MGP-treated cells, suggesting pretranslational activation. GLUT4 was immunodetected intracellularly with a perinuclear predominance. Culture in glucose-free or high-glucose medium did not alter GLUT4 protein content in either control cells or AdCMV-MGP-treated cells. Control and GP-overexpressing cells showed similar autoinhibition of glucose transport, although they appeared to differ in the mechanism(s) involved in this effect. Whereas GLUT1 protein increased in control cells when they were switched from a high-glucose to a glucose-free medium, GLUT1 remained unaltered in GP-expressing cells upon glucose deprivation. Therefore, the increased intracellular metabolic (glycogenolytic-glycolytic) flux that occurs in muscle cells overexpressing GP causes an increase in GLUT4 expression and enhances basal and insulin-stimulated glucose transport, without significant changes in the autoinhibition of glucose transport. This mechanism of regulation may be operative in the postexercise situation in which GLUT4 expression is upregulated in coordination with increased glycolytic flux and energy demand.

Biological Transport↗

PubMed1998

Trafficking pathway of GLUT4 glucose transporters in muscle (review).

Skeletal muscle is a major glucose-utilizing tissue in the absorptive state and alterations in muscle insulin-stimulated glucose uptake lead to derangements in whole body glucose disposal. The major glucose transporter expressed in skeletal muscle is the GLUT4 isoform. In the muscle fiber, GLUT4 undergoes insulin-stimulated translocation to T-tubules and to sarcolemma and it represents a pharmacological target for the treatment of diabetes mellitus, insulin-resistant states or in cardiac dysfunction. We review recent studies describing the characterization of the cellular steps followed by GLUT4 in muscle and we propose several working models for an integrated structure of the GLUT4 trafficking pathway.

Animals↗

PubMed1997

Bcl-2 undergoes phosphorylation by c-Jun N-terminal kinase/stress-activated protein kinases in the presence of the constitutively active GTP-binding protein Rac1.

We have studied the phosphorylation of the Bcl-2 family of proteins by different mitogen-activated protein (MAP) kinases. Purified Bcl-2 was found to be phosphorylated by the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) p54-SAPKbeta, and this is specific insofar as the extracellular signal-regulated kinase 1 (ERK1) and p38/RK/CSBP (p38) catalyzed only weak modification. Bcl-2 undergoes similar phosphorylation in COS-7 when coexpressed together with p54-SAPKbeta and the constitutive Rac1 mutant G12V. This is seen by both 32PO4 labeling and the appearance of five discrete Bcl-2 bands with reduced gel mobility. As anticipated, both intracellular p54-SAPKbeta activation and Bcl-2 phosphorylation are blocked by co-transfection with the MAP kinase specific phosphatase MKP3/PYST1. MAP kinase specificity is also seen in COS-7 cells as Bcl-2 undergoes only weak phosphorylation when co-expressed with enzymatically activated ERK1 or p38. Four critical residues undergoing phosphorylation in COS-7 cells were identified by expression of the quadruple Bcl-2 point mutant T56A,S70A,T74A, S87A. Sequencing phosphopeptides derived from tryptic digests of Bcl-2 indicates that purified GST-p54-SAPKbeta phosphorylates identical sites in vitro. This is the first report of Bcl-2 phosphorylation by the JNK/SAPK class of MAP kinases and could indicate a key modification allowing control of Bcl-2 function by cell surface receptors, Rho family GTPases, and/or cellular stresses.

Amino Acid Sequence↗