Two distinct pathways exist for down-regulation of the TCR

TCR down-regulation plays an important role in modulating T cell responses both during T cell development and in mature T cells. Down-regulation of the TCR is induced by engagement of the TCR by specific ligands and/or by activation of protein kinase C (PKC). We report here that ligand- and PKC-induced TCR down-regulation is mediated by two distinct, independent mechanisms. Ligand-induced TCR down-regulation is dependent on the protein tyrosine kinases p56(lck) and p59(fyn) but independent of PKC and the CD3gamma leucine-based (L-based) internalization motif. In contrast, PKC-induced TCR down-regulation is dependent on the CD3gamma L-based internalization motif but independent of p56(lck) and p59(fyn). Finally, our data indicate that in the absence of TCR ligation, TCR expression levels can be finely regulated via the CD3gamma L-based motif by the balance between PKC and serine/threonine protein phosphatase activities. Such a TCR ligation-independent regulation of TCR expression levels could probably be important in determining the activation threshold of T cells in their encounter with APC.     

The phosphorylation state of CD3gamma influences T cell responsiveness and controls T cell receptor cycling

The T cell receptor (TCR) is internalized following activation of protein kinase C (PKC) via a leucine (Leu)-based motif in CD3gamma. Some studies have indicated that the TCR is recycled back to the cell surface following PKC-mediated internalization. The functional state of recycled TCR and the mechanisms involved in the sorting events following PKC-induced internalization are not known. In this study, we demonstrated that following PKC-induced internalization, the TCR is recycled back to the cell surface in a functional state. TCR recycling was dependent on dephosphorylation of CD3gamma, probably mediated by the serine/threonine protein phosphatase-2A, but independent on microtubules or actin polymerization. Furthermore, in contrast to ligand-mediated TCR sorting, recycling of the TCR was independent of the tyrosine phosphatase CD45 and the Src tyrosine kinases p56(Lck) and p59(Fyn). Studies of mutated TCR and chimeric CD4-CD3gamma molecules demonstrated that CD3gamma did not contain a recycling signal in itself. In contrast, the only sorting information in CD3gamma was the Leu-based motif that mediated lysosomal sorting of chimeric CD4-CD3gamma molecules. Finally, we found a correlation between the phosphorylation state of CD3gamma and T cell responsiveness. Based on these observations a physiological role of CD3gamma and TCR cycling is proposed.     

Substrate-induced conformational change in a trimeric ornithine transcarbamoylase

The crystal structure of Escherichia coli ornithine transcarbamoylase (OTCase, EC 2.1.3.3) complexed with the bisubstrate analog N-(phosphonacetyl)-L-ornithine (PALO) has been determined at 2.8-A resolution. This research on the structure of a transcarbamoylase catalytic trimer with a substrate analog bound provides new insights into the linkages between substrate binding, protein-protein interactions, and conformational change. The structure was solved by molecular replacement with the Pseudomonas aeruginosa catabolic OTCase catalytic trimer (Villeret, V., Tricot, C., Stalon, V. & Dideberg, O. (1995) Proc. Natl. Acad. Sci. USA 92, 10762-10766; Protein Data Bank reference pdb 1otc) as the model and refined to a crystallographic R value of 21.3%. Each polypeptide chain folds into two domains, a carbamoyl phosphate binding domain and an L-ornithine binding domain. The bound inhibitor interacts with the side chains and/or backbone atoms of Lys-53, Ser-55, Thr-56, Arg-57, Thr-58, Arg-106, His-133, Asn-167, Asp-231, Met-236, Leu-274, Arg-319 as well as Gln-82 and Lys-86 from an adjacent chain. Comparison with the unligated P. aeruginosa catabolic OTCase structure indicates that binding of the substrate analog results in closure of the two domains of each chain. As in E. coli aspartate transcarbamoylase, the 240s loop undergoes the largest conformational change upon substrate binding. The clinical implications for human OTCase deficiency are discussed.     

Crystal structures of reaction intermediates of L-2-haloacid dehalogenase and implications for the reaction mechanism

Crystal structures of L-2-haloacid dehalogenase from Pseudomonas sp. YL complexed with monochloroacetate, L-2-chlorobutyrate, L-2-chloro-3-methylbutyrate, or L-2-chloro-4-methylvalerate were determined at 1.83-, 2.0-, 2.2-, and 2.2-A resolutions, respectively, using the complex crystals prepared with the S175A mutant, which are isomorphous with those of the wild-type enzyme. These structures exhibit unique structural features that correspond to those of the reaction intermediates. In each case, the nucleophile Asp-10 is esterified with the dechlorinated moiety of the substrate. The substrate moieties in all but the monochloroacetate intermediate have a D-configuration at the C2 atom. The overall polypeptide fold of each of the intermediates is similar to that of the wild-type enzyme. However, it is clear that the Asp-10-Ser-20 region moves to the active site in all of the intermediates, and the Tyr-91-Asp-102 and Leu-117-Arg-135 regions make conformational changes in all but the monochloroacetate intermediates. Ser-118 is located near the carboxyl group of the substrate moiety; this residue probably serves as a binding residue for the substrate carboxyl group. The hydrophobic pocket, which is primarily composed of the Tyr-12, Gln-42, Leu-45, Phe-60, Lys-151, Asn-177, and Trp-179 side chains, exists around the alkyl group of the substrate moiety. This pocket may play an important role in stabilizing the alkyl group of the substrate moiety through hydrophobic interactions, and may also play a role in determining the stereospecificity of the enzyme. Moreover, a water molecule, which is absent in the substrate-free enzyme, is present in the vicinities of the carboxyl carbon of Asp-10 and the side chains of Asp-180, Asn-177, and Ala-175 in each intermediate. This water molecule may hydrolyze the ester intermediate and its substrate. These findings crystallographically demonstrate that the enzyme reaction proceeds through the formation of an ester intermediate with the enzyme's nucleophile Asp-10.     

Identification of active site residues in the "GyrA" half of yeast DNA topoisomerase II

Site-directed mutagenesis was carried out at 10 highly conserved polar residues within the C-terminal half of yeast DNA topoisomerase II, which corresponds to the A subunit of bacterial DNA gyrase, to identify amino acid side chains that augment the active site tyrosine Tyr-782 in the breakage and rejoining of DNA strands. Complementation tests show that alanine substitution at Arg-690, Asp-697, Lys-700, Arg-704, or Arg-781, but not at His-735, His-736, Glu-738, Gln-750, or Asn-828, inactivates the enzyme in vivo. Measurements of DNA relaxation and cleavage by purified mutant enzymes show that these activities are abolished in the R690A mutant and are much reduced in the mutants D697A, K700A, R704A, and R781A. When a Y782F polypeptide with a phenylalanine substituting for the active site tyrosine was expressed in cells that also express the R690A polypeptide, the resulting heterodimeric yeast DNA topoisomerase II was found to nick plasmid DNA. Thus in a dimeric wild-type enzyme, Tyr-782 in one protomer and Arg-690 in the other cooperate in trans in the catalysis of DNA cleavage. For the residues D697A, K700A, R704A, and R781A, their locations in the crystal structures of type II DNA topoisomerase fragments suggest that Arg-781 and Lys-700 might be involved in anchoring the 5' and 3' sides of the broken DNA, respectively, and the roles of Asp-697 and Arg-704 are probably less direct.     

High affinity binding and allosteric regulation of Escherichia coli glycogen phosphorylase by the histidine phosphocarrier protein, HPr

The histidine phosphocarrier protein (HPr) is an essential element in sugar transport by the bacterial phosphoenolpyruvate:sugar phosphotransferase system. Ligand fishing, using surface plasmon resonance, was used to show the binding of HPr to a nonphosphotransferase protein in extracts of Escherichia coli; the protein was subsequently identified as glycogen phosphorylase (GP). The high affinity (association constant approximately 10(8) M-1), species-specific interaction was also demonstrated in electrophoretic mobility shift experiments by polyacrylamide gel electrophoresis. Equilibrium ultracentrifugation analysis indicates that HPr allosterically regulates the oligomeric state of glycogen phosphorylase. HPr binding increases GP activity to 250% of the level in control assays. Kinetic analysis of coupled enzyme assays shows that the binding of HPr to GP causes a decrease in the Km for glycogen and an increase in the Vmax for phosphate, indicating a mixed type activation. The stimulatory effect of E. coli HPr on E. coli GP activity is species-specific, and the unphosphorylated form of HPr activates GP more than does the phosphorylated form. Replacement of specific amino acids in HPr results in reduced GP activation; HPr residues Arg-17, Lys-24, Lys-27, Lys-40, Ser-46, Gln-51, and Lys-72 were established to be important. This novel mechanism for the regulation of GP provides the first evidence directly linking E. coli HPr to the regulation of carbohydrate metabolism.     

T cell receptor zeta allows stable expression of receptors containing the CD3gamma leucine-based receptor-sorting motif

The leucine-based motif in the T cell receptor (TCR) subunit CD3gamma constitutes a strong internalization signal. In fully assembled TCR this motif is inactive unless phosphorylated. In contrast, the motif is constitutively active in CD4/CD3gamma and Tac/CD3gamma chimeras independently of phosphorylation and leads to rapid internalization and sorting of these chimeras to lysosomal degradation. Because the TCRzeta chain rescues incomplete TCR complexes from lysosomal degradation and allows stable surface expression of fully assembled TCR, we addressed the question whether TCRzeta has the potential to mask the CD3gamma leucine-based motif. By studying CD4/CD3gamma and CD16/CD3gamma chimeras, we found that CD16/CD3gamma chimeras associated with TCRzeta. The CD16/CD3gamma-TCRzeta complexes were stably expressed at the cell surface and had a low spontaneous internalization rate, indicating that the leucine-based motif in these complexes was inactive. In contrast, the CD4/CD3gamma chimeras did not associate with TCRzeta, and the leucine-based motif in these chimeras was constitutively active resulting in a high spontaneous internalization rate and low expression of the chimeras at the cell surface. Thus, our data demonstrate that TCRzeta allows stable cell surface expression of receptors containing CD3gamma leucine-based motifs by its potential to mask such motifs.     

Cysteine-scanning mutagenesis around transmembrane segment III of Tn10-encoded metal-tetracycline/H+ antiporter

Each amino acid in the putative transmembrane helix III and its flanking regions (from Gly-62 to Tyr-98) of the Tn10-encoded metal-tetracycline/H+ antiporter (Tet(B)) was individually replaced with Cys. Out of these 37 cysteine-scanning mutants, the mutants from G62C to R70C and from S92C to Y98C showed high or intermediate reactivity with [14C]N-ethylmaleimide (NEM) except for the M64C mutant. On the other hand, the mutants from R71C to S91C showed almost no reactivity with NEM except for the P72C mutant. These results confirm that the transmembrane helix III is composed of 21 residues from Arg-71 to Ser-91. The majority of Cys replacement mutants retained high or moderate tetracycline transport activity. Cys replacements for Gly-62, Asp-66, Ser-77, Gly-80, and Asp-84 resulted in almost inactive Tet(B) (less than 3% of the wild-type activity). The Arg-70 --> Cys mutant retained very low activity due to a mercaptide between Co2+ and a SH group (Someya, Y., and Yamaguchi, A. (1996) Biochemistry 35, 9385-9391). Three of these six important residues (Ser-77, Gly-80, and Asp-84) are located in the transmembrane helix III and one (Arg-70) is located in the flanking region. These four functionally important residues are located on one side of the helical wheel. Only two of the residual 31 Cys mutants were inactivated by NEM (S65C and L97C). Ser-65 and Leu-97 are located on the cytoplasmic and periplasmic loops, respectively, in the topology of Tet(B). The degree of inactivation of these Cys mutants with SH reagents was dependent on the volume of substituents. In the presence of tetracycline, the reactivity of the S65C mutant with NEM was significantly increased, in contrast, the reactivity of L97C was greatly reduced, indicating that the cytoplasmic and periplasmic loop regions undergo substrate-induced conformational change in the mutually opposite direction.     

Mechanism of DNA transesterification by vaccinia topoisomerase: catalytic contributions of essential residues Arg-130, Gly-132, Tyr-136 and Lys-167

Vaccinia topoisomerase, a eukaryotic type IB enzyme, catalyzes relaxation of supercoiled DNA by cleaving and rejoining DNA strands through a DNA- (3'-phosphotyrosyl)-enzyme intermediate. We have performed a kinetic analysis of mutational effects at four essential amino acids: Arg-130, Gly-132, Tyr-136 and Lys-167. Arg-130, Gly-132 and Lys-167 are conserved in all members of the type IB topoisomerase family. Tyr-136 is conserved in all poxvirus topoisomerases. We show that Arg-130 and Lys-167 are required for transesterification chemistry. Arg-130 enhances the rates of both cleavage and religation by 10(5). Lys-167 enhances the cleavage and religation reactions by 10(3) and 10(4), respectively. An instructive distinction between these two essential residues is that Arg-130 cannot be replaced by lysine, whereas substituting Lys-167 by arginine resulted in partial restoration of function relative to the alanine mutant. We propose that both basic residues interact directly with the scissile phosphate at the topoisomerase active site. Mutations at positions Gly-132 and Tyr-136 reduced the rate of strand cleavage by more than two orders of magnitude, but elicited only mild effects on religation rate. Gly-132 and Tyr-136 are suggested to facilitate a pre-cleavage activation step. The results of comprehensive mutagenesis of the vaccinia topoisomerase illuminate mechanistic and structural similarities to site-specific recombinases.     

Mapping a telomere using the translocation eT1(III;V) in Caenorhabditis elegans

TCR engagement activates phospholipase C gamma 1 (PLC gamma 1) via a tyrosine phosphorylation-dependent mechanism. PLC gamma 1 contains a pair of Src homology 2 (SH2) domains whose function is that of promoting protein interactions by binding phosphorylated tyrosine and adjacent amino acids. The role of the PLC gamma 1 SH2 domains in PLC gamma 1 phosphorylation was explored by mutational analysis of an epitope-tagged protein transiently expressed in Jurkat T cells. Mutation of the amino-terminal SH2 domain (SH2(N) domain) resulted in defective tyrosine phosphorylation of PLC gamma 1 in response to TCR/CD3 perturbation. In addition, the PLC gamma 1 SH2(N) domain mutant failed to associate with Grb2 and a 36- to 38-kDa phosphoprotein (p36-38), which has previously been recognized to interact with PLC gamma 1, Grb2, and other molecules involved in TCR signal transduction. Conversely, mutation of the carboxyl-terminal SH2 domain (SH2(C) domain) did not affect TCR-induced tyrosine phosphorylation of PLC gamma 1. Furthermore, binding of p36-38 to PLC gamma 1 was not abrogated by mutations of the SH2(C) domain. In contrast to TCR/CD3 ligation, treatment of cells with pervanadate induced tyrosine phosphorylation of either PLC gamma 1 SH2(N) or SH2(C) domain mutants to a level comparable with that of the wild-type protein, indicating that pervanadate treatment induces an alternate mechanism of PLC gamma 1 phosphorylation. These data indicate that the SH2(N) domain is required for TCR-induced PLC gamma 1 phosphorylation, presumably by participating in the formation of a complex that promotes the association of PLC gamma 1 with a tyrosine kinase.     

Differential regulation of cardiac actomyosin S-1 MgATPase by protein kinase C isozyme-specific phosphorylation of specific sites in cardiac troponin I and its phosphorylation site mutants

The significance of site-specific phosphorylation by protein kinase C (PKC) isozymes alpha and delta and protein kinase A (PKA) of troponin I (TnI) and its phosphorylation site mutants in the regulation of Ca(2+)-stimulated MgATPase activity of reconstituted actomyosin S-1 was investigated. The genetically defined TnI mutants used were T144A, S43A/S45A, S43A/S45A/T144A (in which the PKC phosphorylation sites Thr-144 and Ser-43/Ser-45 were respectively substituted by Ala) and N32 (in which the first 32 amino acids in the NH2-terminal sequence containing Ser-23/Ser-24 were deleted). Although the PKC isozymes displayed different substrate phosphorylation kinetics, PKC-alpha phosphorylated equally well TnI wild type and all mutants, whereas N32 was a much poorer substrate for PKC-delta. Furthermore, the two PKC isozymes exhibited discrete specificities in phosphorylating distinct sites in TnI and its mutants, either as individual subunits or as components of the reconstituted troponin complex. Unlike PKC-alpha, PKC-delta favorably phosphorylated the PKA-preferred site Ser-23/Ser-24 and hence, like PKA, reduced the Ca2+ sensitivity of the reconstituted actomyosin S-1 MgATPase. In contrast, PKC-alpha preferred to phosphorylate Ser-43/Ser-45 (common sites for all isozymes) and thus reduced the maximal Ca(2+)-stimulated activity of the MgATPase. In this respect, PKC-delta, by cross-phosphorylating the PKA sites, functioned as a hybrid of PKC-alpha and PKA. The site specificities and hence functional differences between PKC-alpha and -delta were most evident at low phosphorylation (1 mol of phosphate/mol) of TnI wild type and were magnified when S43A/S45A and N32 were used as substrates. The present study has demonstrated, for the first time, that distinct functional consequences could arise from the site-selective preferences of PKC-alpha and -delta for phosphorylating a single substrate in the myocardium, i.e., TnI.     

Protein kinase C-dependent regulation of Na+/Ca2+ exchanger isoforms NCX1 and NCX3 does not require their direct phosphorylation

We compared the phosphorylation-dependent regulation of three mammalian Na+/Ca2+ exchanger isoforms (NCX1-NCX3) expressed in CCL39 fibroblasts that have little endogenous activity. Na+i-dependent 45Ca2+ uptake into NCX1- or NCX3-expressing cells, but not that into NCX2-expressing cells, was significantly enhanced by phorbol 12-myristate 13-acetate (PMA) or platelet-derived growth factor-BB, which was abolished by pretreatment of cells with calphostin C or a prior long exposure to PMA. This suggests that NCX1 or NCX3, but not NCX2, is stimulated by a pathway involving protein kinase C (PKC). Immunoprecipitation experiments using [32P]orthophosphate-labeled cells revealed that both NCX2 and NCX3 proteins were phosphorylated to a much lesser extent than the NCX1 protein in unstimulated cells and that the extent of phosphorylation was not increased by treatment with PKC activators, although NCX1 phosphorylation was enhanced significantly. Using site-directed mutagenesis, we identified three phosphorylation sites in the NCX1 protein in the PMA-stimulated cells to be Ser-249, Ser-250, and Ser-357 with Ser-250 being predominantly phosphorylated. We found that the NCX1 mutant with these serine residues substituted with alanine still maintained a normal response to PMA. In contrast, the NCX1 or NCX3 mutant, with the large central cytoplasmic loop deleted, lost the responsiveness to PMA. These results suggest that the PKC-dependent regulation of NCX1 or NCX3 requires the central cytoplasmic loop but does not require the direct phosphorylation of the exchanger.     

FK506 binding protein mutational analysis. Defining the surface residue contributions to stability of the calcineurin co-complex

The 12- and 13-kDa FK506 binding proteins (FKBP12 and FKBP13) are cis-trans peptidyl-prolyl isomerases that bind the macrolides FK506 (Tacrolimus) and rapamycin (Sirolimus). The FKBP12.FK506 complex is immunosuppressive, acting as an inhibitor of the protein phosphatase calcineurin. We have examined the role of the key surface residues of FKBP12 and FKBP13 in calcineurin interactions by generating substitutions at these residues by site-directed mutagenesis. All mutants are active catalysts of the prolyl isomerase reaction, and bind FK506 or rapamycin with high affinity. Mutations at FKBP12 residues Asp-37, Arg-42, His-87, and Ile-90 decrease calcineurin affinity of the mutant FKBP12.FK506 complex by as much as 2600-fold in the case of I90K. Replacement of three FKBP13 surface residues (Gln-50, Ala-95, and Lys-98) with the corresponding homologous FKBP12 residues (Arg-42, His-87, and Ile-90) generates an FKBP13 variant that is equivalent to FKBP12 in its affinity for FK506, rapamycin, and calcineurin. These results confirm the role of two loop regions of FKBP12 (residues 40-44 and 84-91) as part of the effector face that interacts with calcineurin.     

Differential cytosolic tail dependence and intracellular fate of T-cell receptors internalized upon activation with superantigen or phorbol ester

Activation of T lymphocytes by T-cell receptor (TCR) ligands such as peptide/MHC complexes, superantigens or anti-TCR mAbs, or by pharmacological activators of protein kinase C such as phorbol esters, results in the internalization and cell surface downregulation of TCRs. We investigated the role of internalization motifs located in the cytosolic region of CD3 gamma in the internalization of TCR complexes induced by enterotoxin superantigens, anti-TCR mAbs or phorbol esters. To this end, a series of CD3 gamma mutants were expressed in a CD3 gamma-deficient variant of the human T-cell line Jurkat. We found that serine126 and the di-leucine motif (Leu131-Leu132) are required for phorbol-ester-induced TCR downregulation, but they are not necessary for enterotoxin superantigen or antibody-induced TCR downregulation. Moreover, the tyrosine-based motifs (residues 138 to 141 and 149 to 152) are not required either for phorbol aster or for superantigen or antibody-induced TCR downregulation. Confocal microscopy analysis reveals that TCR complexes accumulate in an early endocytic/recycling compartment upon activation of cells with phorbol esters, whereas TCRs internalized upon activation with superantigen or anti-TCR mAbs are routed to lysosomes. Consistent with this intracellular localization, TCRs internalized in response to phorbol ester are not degraded and can be reexpressed on the cell surface. In contrast, TCRs internalized upon superantigen activation are degraded.     

Protein kinase C-mediated regulation of L-type Ca channels from skeletal muscle requires phosphorylation of the alpha 1 subunit

Dihydropyridine-sensitive, L-type Ca channels are hetero-oligomeric proteins that are modulated in certain cell types by protein kinase C (PKC). In native skeletal muscle membranes, PKC phosphorylates the alpha 1 and beta subunits of these Ca channels and modulates channel activity. However, it is unknown if phosphorylation of both subunits is necessary for PKC-mediated channel regulation. Here we report that stoichiometric phosphorylation of the alpha 1 subunit was required for activation of these Ca channels by PKC, while PKC-mediated phosphorylation of the beta subunit alone did not modify channel activity. Furthermore, reversal of the functional effects of PKC by protein phosphatase-1c was quantitatively correlated with dephosphorylation of the alpha 1 subunit.     

Specificity of an extracellular proteinase from Brevibacterium linens ATCC 9174 on bovine alpha s1-casein

The specificity of the extracellular proteinase from Brevibacterium linens ATCC 9174 on bovine alpha s1-casein was studied. Hydrolysis was monitored over time by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) and urea-PAGE. The major pH 4.6-soluble peptides were isolated by high-performance liquid chromatography and identified by N-terminal amino acid sequencing and mass spectrometry. The time course of peptide formation indicated that His-8-Gln-9, Ser-161-Gly-162, and either Gln-172-Tyr-173 or Phe-23-Phe-24 were the first, second, and third bonds cleaved, respectively. Other cleavage sites included Asn-19-Leu-20, Phe-32-Gly-33, Tyr-104-Lys-105, Leu-142-Ala-143, Phe-150-Arg-151, Gln-152-Phe-153, Leu-169-Gly-170, and Thr-171-Gln-172. The proteinase had a broad specificity for the amino acid residues at the P1 and P'1 positions but showed a preference for hydrophobic residues at the P2, P3, P4, P'2, P'3, and P'4 positions.     

Distribution and developmental regulation of metabotropic glutamate receptor 7a in rat brain

Ligation of the TCR-CD3 complex initiates a cascade of tyrosine phosphorylation that results in T cell activation. Initial activation of tyrosine kinases depends on the phosphorylation of activation motifs on CD3 chains. We previously found that a 90-kDa protein was tyrosine phosphorylated upon TCR cross-linking and the induction of the phosphorylation was dependent on the structure of the CD3 complex. In this study, we further characterized p90 phosphorylation. Phosphorylation of p90 was induced only by stimulation through the TCR-CD3 complex but not by other kinds of stimulation including CD28- or hydrogen peroxide-mediated activation and was dynamically regulated. Phosphorylated p90 was associated with the TCR-CD3 complex upon T cell activation. In a normal T cell population, thymocytes but not splenic T cells induced the tyrosine phosphorylation of p90 upon TCR cross-linking. These results suggest that p90 is a novel phosphoprotein associated with the TCR-CD3 complex and may play a role in TCR signaling during thymocyte differentiation.