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Eukaryotic translation initiation factor 2B (eIF2B) is the heteropentameric guanine nucleotide exchange factor for translation initiation factor 2 (eIF2). Recent studies in the yeast Saccharomyces cerevisiae have served to characterize genetically the exchange factor. However, enzyme kinetic studies of the yeast enzyme have been hindered by the lack of sufficient quantities of protein suitable for biochemical analysis. We have purified yeast eIF2B and characterized its catalytic properties in vitro. Values for K(m) and V(max) were determined to be 12.2 nm and 250.7 fmol/min, respectively, at 0 degrees C. The calculated turnover number (K(cat)) of 43.2 pmol of GDP released per min/pmol of eIF2B at 30 degrees C is approximately 1 order of magnitude lower than values previously reported for the mammalian factor. Reciprocal plots at varying fixed concentrations of the second substrate were linear and intersected to the left of the y axis. This is consistent with a sequential catalytic mechanism and argues against a ping-pong mechanism similar to that proposed for EF-Tu/EF-Ts. In support of this model, our yeast eIF2B preparations bind guanine nucleotides, with an apparent dissociation constant for GTP in the low micromolar range.

The new member of the Ras superfamily of G-proteins, Rheb, has been identified in rat and human, but its function has not been defined. We report here the identification of Rheb homologues in the budding yeast Saccharomyces cerevisiae (ScRheb) as well as in Schizosaccharomyces pombe, Drosophila melanogaster, zebrafish, and Ciona intestinalis. These proteins define a new class of G-proteins based on 1) their overall sequence similarity, 2) high conservation of their effector domain sequence, 3) presence of a unique arginine in their G1 box, and 4) presence of a conserved CAAX farnesylation motif. Characterization of an S. cerevisiae strain deficient in ScRheb showed that it is hypersensitive to growth inhibitory effects of canavanine and thialysine, which are analogues of arginine and lysine, respectively. Accordingly, the uptake of arginine and lysine was increased in the ScRheb-deficient strain. This increased arginine uptake requires the arginine-specific permease Can1p. The function of ScRheb is dependent on having an intact effector domain since mutations in the effector domain of ScRheb are incapable of complementing canavanine hypersensitivity of scrheb disruptant cells. Furthermore, the conserved arginine in the G1 box plays a role in the activity of ScRheb, as a mutation of this arginine to glycine significantly reduced the ability of ScRheb to complement canavanine hypersensitivity of ScRheb-deficient yeast. Finally, a mutation in the C-terminal CAAX farnesylation motif resulted in a loss of ScRheb function. This result, in combination with our finding that ScRheb is farnesylated, suggests that farnesylation plays a key role in ScRheb function. Our findings assign the regulation of arginine and lysine uptake as the first physiological function for this new farnesylated Ras superfamily G-protein.

Protein farnesyltransferase (FTase) plays important roles in the growth and differentiation of eukaryotic cells. In this paper, we report the identification of the Schizosaccharomyces pombe gene cpp1(+) encoding the beta-subunit of FTase. The predicted amino acid sequence of the cpp1(+) gene product shares significant similarity with FTase beta-subunits from a variety of organisms. S. pombe FTase purified from E. coli exhibits high enzymatic activity toward the CAAX farnesylation motif substrates (where C represents cysteine, A represents aliphatic amino acid, and X is preferentially methionine, cysteine, serine, alanine, or glutamine) while showing little preference for CAAL geranylgeranylation motif substrates (where L represents leucine or phenylalanine). cpp1(+) is not essential for growth as shown by gene disruption; however, mutant cells exhibit rounded or irregular cell morphology. Expression of a geranylgeranylated mutant form, Ras1-CVIL, which can bypass farnesylation, rescues these morphological defects. We also identify a novel phenotype of cpp1(-) mutants, hypersensitivity to canavanine. This appears to be due to a 3-4-fold increase in the rate of arginine uptake as compared with wild-type cells. Expression of the geranylgeranylated mutant form of a novel farnesylated small GTPase, SpRheb, is able to suppress the elevated arginine uptake rate. These results demonstrate that protein farnesylation is critical for maintaining normal cell morphology through Ras1 and canavanine resistance through SpRheb.

A novel human organic transporter, OATP2, has been identified that transports taurocholic acid, the adrenal androgen dehydroepiandrosterone sulfate, and thyroid hormone, as well as the hydroxymethylglutaryl-CoA reductase inhibitor, pravastatin. OATP2 is expressed exclusively in liver in contrast to all other known transporter subtypes that are found in both hepatic and nonhepatic tissues. OATP2 is considerably diverged from other family members, sharing only 42% sequence identity with the four other subtypes. Furthermore, unlike other subtypes, OATP2 did not transport digoxin or aldosterone. The rat isoform oatp1 was also shown to transport pravastatin, whereas other members of the OATP family, i.e. rat oatp2, human OATP, and the prostaglandin transporter, did not. Cis-inhibition studies indicate that both OATP2 and roatp1 also transport other statins including lovastatin, simvastatin, and atorvastatin. In summary, OATP2 is a novel organic anion transport protein that has overlapping but not identical substrate specificities with each of the other subtypes and, with its liver-specific expression, represents a functionally distinct OATP isoform. Furthermore, the identification of oatp1 and OATP2 as pravastatin transporters suggests that they are responsible for the hepatic uptake of this liver-specific hydroxymethylglutaryl-CoA reductase inhibitor in rat and man.

CXCR2 is a seven-transmembrane receptor that transduces intracellular signals in response to the chemokines interleukin-8, melanoma growth-stimulatory activity/growth-regulatory protein, and other ELR motif-containing CXC chemokines by coupling to heterotrimeric GTP-binding proteins. In this study, we explored the mechanism responsible for ligand-induced CXCR2 endocytosis. Here, we demonstrate that dynamin, a component of clathrin-mediated endocytosis, is essential for CXCR2 endocytosis and resensitization. In HEK293 cells, dynamin I K44A, a dominant-negative mutant of dynamin that inhibits the clathrin-mediated endocytosis, blocks the ligand-stimulated CXCR2 sequestration. Furthermore, co-expression of dynamin I K44A significantly delays dephosphorylation of CXCR2 after ligand stimulation, suggesting that clathrin-mediated endocytosis plays an important role in receptor dephosphorylation and resensitization. In addition, ligand-mediated receptor down-regulation is attenuated when receptor internalization is inhibited by dynamin I K44A. Interestingly, inhibition of receptor endocytosis by dynamin I K44A does not affect the CXCR2-mediated stimulation of mitogen-activated protein kinase. Most significantly, our data indicate that the ligand-stimulated receptor endocytosis is required for CXCR2-mediated chemotaxis in HEK293 cells. Taken together, our findings suggest that clathrin-mediated CXCR2 internalization is crucial for receptor endocytosis, resensitization, and chemotaxis.

The MutS DNA mismatch protein recognizes heteroduplex DNAs containing mispaired or unpaired bases. We have examined the oligomerization of a MutS protein from Thermus aquaticus that binds to heteroduplex DNAs at elevated temperatures. Analytical gel filtration, cross-linking of MutS protein with disuccinimidyl suberate, light scattering, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry establish that the Taq protein is largely a dimer in free solution. Analytical equilibrium sedimentation showed that the oligomerization of Taq MutS involves a dimer-tetramer equilibrium in which dimer predominates at concentrations below 10 microM. The DeltaG(0)(2-4) for the dimer to tetramer transition is approximately -6.9 +/- 0.1 kcal/mol of tetramer. Analytical gel filtration of native complexes and gel mobility shift assays of an maltose-binding protein-MutS fusion protein bound to a short, 37-base pair heteroduplex DNA reveal that the protein binds to DNA as a dimer with no change in oligomerization upon DNA binding.

The transcriptional regulation of the apoCIII gene by hormonal and metabolic signals plays a significant role in determining plasma triglyceride levels. In the current work we demonstrate that the apoCIII gene is regulated by the mitogen-activated protein (MAP) kinase signaling pathway. In HepG2 cells, repression of MAP kinase activity by treatment with the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibitor PD98059 caused a 5-8-fold increase in apoCIII transcriptional activity. Activation of MAP kinase by phorbol ester treatment caused a 3-5-fold reduction in apoCIII transcription. The region of the apoCIII promoter responsible for this regulation was mapped in transiently transfected HepG2 cells to a 6-base pair element located at -740. The major protein binding to this site was identified as the nuclear hormone receptor HNF4. An increase in HNF4 mRNA and protein levels was observed in HepG2 cells after treatment with PD98059, indicating that the MAP kinase pathway regulates the expression of the HNF4 gene. These findings demonstrate that the apoCIII gene can be regulated by signals acting through the MAP kinase pathway and that this regulation is mediated, at least in part, by changes in the amount of HNF4.

Activated Cdc42-associated kinase-2 (ACK-2) is a non-receptor tyrosine kinase that appears to be a highly specific target for the Rho-related GTP-binding protein Cdc42. In order to understand better how ACK-2 activity is regulated in cells, we have expressed epitope-tagged forms of this tyrosine kinase in COS-7 and NIH3T3 cells. We find that ACK-2 can be activated by cell adhesion in a Cdc42-dependent manner. However, unlike the focal adhesion kinase, which also is activated by cell adhesion, the activation of ACK-2 is F-actin-independent and does not require cell spreading. In addition, overexpression of ACK-2 in COS-7 cells did not result in the stimulation of extracellular signal-regulated kinase activity but rather activated the c-Jun kinase. Both anti-integrin beta1 antibody and RGD peptides inhibited the activation of ACK-2 by cell adhesion. In addition, ACK-2 was co-immunoprecipitated with integrin beta1. Overall, these findings suggest that ACK-2 interacts with integrin complexes and mediates cell adhesion signals in a Cdc42-dependent manner.

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) plays a major role in the stimulation of bone growth, mineralization, and intestinal calcium and phosphate absorption; it also acts as a general inhibitor of cellular proliferation. Several new, clinically relevant compounds dissociate antiproliferative and calcemic activities of 1,25(OH)2D3, but the molecular basis for this has not been clearly elucidated. Here, we tested whether the potency of one class of compounds, 20-epi analogues, to induce myeloid cell differentiation, is because of direct molecular effects on vitamin D receptor (VDR). We report that two 20-epi analogues, MC1627 and MC1288, induced differentiation and transcription of p21(Waf1,Cip1), a key VDR target gene involved in growth inhibition, at a concentration 100-fold lower than that of 1,25(OH)2D3. We compared this sensitivity to analogue effects on VDR interacting proteins: RXR, GRIP-1, and DRIP205, a subunit of the DRIP coactivator complex. Compared with the interaction of VDR with RXR or GRIP-1, the differentiation dose-response most closely correlated to the ligand-dependent recruitment of the DRIP coactivator complex to VDR and to the ability of the receptor to activate transcription in a cell-free system. These results provide compelling links between the efficiency of the 20-epi analogue in inducing VDR/DRIP interactions, transactivation in vitro, and its enhanced ability to induce cellular differentiation.

The Tec protein-tyrosine kinase family includes Btk, Itk/Tsk/Emt, Tec, Rlk/Txk, and Bmx which are involved in signals mediated by various cytokines or antigen receptors. Itk is expressed primarily in T cells and activated by TCR/CD3, CD28, and CD2. However, the defect in T cell signaling in itk-deficient mice is very modest. Thus, we looked for other Tec family kinases that could be expressed in lymphoid cells and involved in T cell signal transduction. Here, we demonstrate that Tec, expressed in T cells, is activated following TCR/CD3 or CD28 ligation and interacts with CD28 receptor in an activation-dependent manner. This interaction involves the Tec SH3 domain and the proline-rich motifs in CD28. We also show that Tec can phosphorylate p62(dok), one CD28-specific substrate, whereas Itk cannot. Overexpression of Tec but not Itk can enhance the interleukin-2 promoter activity mediated by TCR/CD3 or CD28 stimulation and introduction of a kinase-dead Tec but not Itk can suppress interleukin-2 expression, indicating that Tec is directly involved in T cell activation. Altogether, these data demonstrate that Tec kinase is an integral component of T cell signaling and that the two Tec family kinases, Tec and Itk, have distinct roles in T cell activation.

Mutations in Bruton's tyrosine kinase (Btk) have been associated with immunodeficiencies in man and in the mouse. Btk and two related proteins, Itk and Tec, are members of a distinct family of tyrosine kinases. These kinases are believed to function in various receptor-mediated signaling pathways, but their specific functions are as yet undefined. Btk and its homologues share extensive sequence similarity, including a conserved region, the Tec-homology (TH) domain, that has been proposed to mediate specific intermolecular or intramolecular interactions. The TH region of Btk contains a functional SH3-binding site at residues 189-192. SH3 binding is selective: Btk is retained by the SH3 domain of Fyn but not by that of Blk, another Src-type kinase. TH-SH3 binding in vitro is abolished by specific, single amino acid substitutions within the Btk TH domain or the Fyn SH3 domain. We provide two lines of evidence that the SH3-binding site in the Btk TH domain mediates protein interactions in intact cells. First, treatment of cells with pervanadate induces an increase in the phosphotyrosine content of kinase-inactive Btk; this response is substantially reduced by a mutation that inactivates the SH3-binding site in the Btk TH domain. Second, in cell lysates Btk is found in association with an as yet unidentified 72-kDa phosphotyrosine-containing protein; this interaction requires a functional SH3-binding site in the TH domain. The TH domain may therefore interact in vivo with other proteins that regulate the phosphorylation state of Btk.

Mitochondrial creatine kinase was first proposed to act as a functional component in respiratory control in 1966 (Bessman, S. P., and Fonyo, A. (1966) Biochem. Biophys. Res. Commun. 22, 597-602). Since that time, evidence has accumulated to support the theory of a creatine-phosphorylcreatine shuttle mechanism involved in supplying energy for aerobic muscle contraction (Bessman, S. P., and Geiger, P. J. (1981) Science 211, 448-452). To demonstrate directly the interaction between mitochondrial oxidative phosphorylation and that of creatine phosphate synthesis, we have studied the labeling of adenine nucleotides and creatine phosphate with [33P]H3PO4 or [gamma-32P]ATP over a range of adenine nucleotide concentrations incubated with rabbit cardiac and rat skeletal muscle mitochondria. An apparent direct mitochondrial ATP contribution to creatine phosphate synthesis was observed that varied inversely with the total adenine nucleotide present in the reaction system. This reaction of de novo synthesized ATP with creatine phosphokinase prior to equilibration with the total ATP pool was observed regardless of the entry point of electrons from oxidizable substrate into the electron transport chain. This special relation was not observed for added yeast hexokinase in forming glucose 6-phosphate. Mitochondria could not synthesize creatine phosphate in the presence of atractyloside, thus underscoring the requirement for adenine nucleotide translocase-linked transport of ATP prior to reaction with the bound creatine phosphokinase. These studies show that there is coupling or compartmentation of ATP synthesis and transport with creatine phosphate formation in heart and skeletal muscle mitochondria.

Amiloride is a reversible inhibitor of the Na+/H+ antiporter which acts at the external aspect of the transport system. The kinetics of inhibition of the Na+/H+ antiporter with amiloride have been controversial, with the usual finding of simple competitive inhibition, but with other reports of mixed and noncompetitive inhibition of the transporter by amiloride. The present experiments demonstrate that the chloride content of the external transport buffer affects the kinetics of amiloride inhibition. Either simple competitive or mixed inhibition by amiloride was observed in the same vesicle preparations depending on the presence of chloride or gluconate in the buffer. The effect of chloride on the inhibitory effect of amiloride was dependent on the concentration of chloride and amiloride. Similar effects were observed with more potent analogues of amiloride. These findings suggest that the external aspect of the antiporter has a site or sites at which the inhibitory effects of amiloride on the Na+/H+ antiporter can be modified by chloride, even though chloride has only slight effects on the kinetics of the Na+/H+ antiporter in the absence of amiloride.

The nucleotide-dependent tetramerization of purified native URA7-encoded CTP synthetase (EC 6.3.4.2, UTP: ammonia ligase (ADP-forming)) from the yeast Saccharomyces cerevisiae was characterized. CTP synthetase existed as a dimer in the absence of ATP and UTP. In the presence of saturating concentrations of ATP and UTP, the CTP synthetase protein existed as a tetramer. Increasing concentrations of ATP and UTP caused a dose-dependent conversion of the dimeric species to a tetramer. The kinetics of enzyme tetramerization correlates with the kinetics of enzyme activity. The tetramerization of CTP synthetase was dependent on UTP and Mg2+ ions. ATP facilitated the UTP-dependent tetramerization of CTP synthetase by a mechanism that involved the ATP-dependent phosphorylation of UTP catalyzed by the enzyme. The glutaminase reaction that is catalyzed by the enzyme was not required for enzyme tetramerization. CTP, a potent inhibitor of CTP synthetase activity, did not inhibit the ATP/UTP-dependent tetramerization of the enzyme. Phosphorylation of the purified native CTP synthetase with protein kinase A and protein kinase C facilitated the nucleotide-dependent tetramerization. Dephosphorylation of native CTP synthetase with alkaline phosphatase prevented the nucleotide-dependent tetramerization of the enzyme. This correlated with the inactivation of CTP synthetase activity. Rephosphorylation of the dephosphorylated enzyme with protein kinase A and protein kinase C resulted in a partial restoration of the nucleotide-dependent tetramerization of the enzyme. This tetramerization correlated with the partial restoration of CTP synthetase activity. Taken together, these results indicated that enzyme tetramerization was required for CTP synthetase activity and that enzyme phosphorylation played an important role in the tetramerization and regulation of the enzyme.

A set of host-guest peptides of the form Ac(Gly-Pro-Hyp)3-Gly-X-Y-(Gly-Pro-Hyp)4-Gly-Gly-NH2 has been designed to evaluate the propensity of different Gly-X-Y triplets for the triple-helix conformation (Shah, N. K., Ramshaw, J. A. M., Kirkpatrick, A., Shah, C., and Brodsky, B. (1996) Biochemistry 35, 10262-10268). All Gly-X-Y guest triplets led to a decrease in melting temperature from the host (Gly-Pro-Hyp)8 peptide except for Gly-Pro-Arg. In this Gly-Pro-Hyp-rich environment, Gly-Pro-Arg was found to be as stabilizing as Gly-Pro-Hyp. Decreased stability of host-guest peptides containing Gly-Pro-Lys, Gly-Pro-homo-Arg, and Gly-Arg-Hyp compared with Gly-Pro-Arg indicated a stabilization that is optimal for Arg and specific to the Y-position. Arg was found to have a similar stabilizing effect when residues other than Pro are in the X-position. Both Arg and Hyp stabilize the triple-helix preferentially in the Y-position in a stereospecific manner and occupy largely Y-positions in collagen. However, contiguous Gly-Pro-Hyp units are highly stable and promote triple-helix folding, whereas incorporation of multiple Gly-Pro-Arg triplets was destabilizing and folded slowly due to charge repulsion. In collagen, Gly-Pro-Arg may contribute maximally to local triple-helix stability while also having the potential for electrostatic interactions in fibril formation and binding.

The hepatitis B virus X protein induces transcriptional activation of a wide variety of viral and cellular genes. In addition to its ability to interact directly with many nuclear transcription factors, several reports indicate that the X protein stimulates different cytoplasmic kinase signal cascades. Using the yeast two-hybrid screen, we have isolated a clone designated X-associated protein 3 (XAP3) that encodes a human homolog of the rat protein kinase C-binding protein. One of the activation domains of X (amino acids 90-122) is required for binding to XAP3, while the NH2-terminal part of XAP3 is necessary for binding to X. Both X and XAP3 bound specifically to the eta PKC isoenzyme synthesized in rabbit reticulocyte lysates. Overexpression of XAP3 enhanced X transactivation activity. These results support earlier findings that one of the mechanisms of transactivation by X is through involvement with the cellular protein kinase C pathway.

CTP synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)) is an allosterically regulated enzyme in the yeast Saccharomyces cerevisiae. In this work we examined the regulation of CTP synthetase activity by S. cerevisiae protein kinase C (Pkc1p) phosphorylation. The results of labeling experiments with S. cerevisiae mutants expressing different levels of the PKC1 gene indicated that phosphorylation of CTP synthetase was mediated by Pkc1p in vivo. In vitro, Pkc1p phosphorylated purified CTP synthetase on serine and threonine residues, which resulted in the activation (3-fold) of enzyme activity. The mechanism of this activation involved an increase in the apparent Vmax of the reaction and an increase in the enzyme's affinity for ATP. In vitro phosphorylated CTP synthetase also exhibited a decrease in its positive cooperative kinetic behavior with respect to UTP and ATP. Phosphorylation of CTP synthetase did not have a significant effect on the kinetic properties of the enzyme with respect to glutamine and GTP. Phosphorylation of CTP synthetase resulted in a decrease in the enzyme's sensitivity to product inhibition by CTP. Phosphorylation did not affect the mechanism by which CTP inhibits CTP synthetase activity.

Sialyl-Lex (sLex) antigen expression recognized by KM93 monoclonal antibody was significantly down-regulated during differentiation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in human pre-B lymphocytic leukemia cell line KM3. The sLex determinants were almost exclusively expressed on O-linked oligosaccharide chains of an O-glycosylated 150-kDa glycoprotein (gp150). A low shear force cell adhesion assay showed that TPA treatment significantly inhibited E-selectin-mediated cell adhesion. Transcript and/or enzyme activity levels of alpha1-->3-fucosyltransferase, alpha2-->3-sialyltransferase, beta1-->4-galactosyltransferase, and elongation beta1-->3-N-acetylglucosaminyltransferase did not correlate with sLex expression levels. However, transcript and enzyme activity levels of core 2 GlcNAc-transferase (C2GnT) were significantly down-regulated during TPA treatment. Following transfection and constitutive expression of full-length exogenous C2GnT transcript, C2GnT enzyme activities were maintained at high levels even after TPA treatment and down-regulation of cell surface sLex antigen expression by TPA was completely abolished. Furthermore, in the transfected cells, the KM93 reactivity of gp150 was not reduced by TPA treatment, and the inhibition of cell adhesion by TPA was also blocked. These results suggest that sLex expression is critically regulated by a single glycosyltransferase, C2GnT, during differentiation of KM3 cells.

Benign familial neonatal convulsions (BFNC), a class of idiopathic generalized epilepsy, is an autosomal dominantly inherited disorder of newborns. BFNC has been linked to mutations in two putative K+ channel genes, KCNQ2 and KCNQ3. Amino acid sequence comparison reveals that both genes share strong homology to KvLQT1, the potassium channel encoded by KCNQ1, which is responsible for over 50% of inherited long QT syndrome. Here we describe the cloning, functional expression, and characterization of K+ channels encoded by KCNQ2 and KCNQ3 cDNAs. Individually, expression of KCNQ2 or KCNQ3 in Xenopus oocytes elicits voltage-gated, rapidly activating K+-selective currents similar to KCNQ1. However, unlike KCNQ1, KCNQ2 and KCNQ3 currents are not augmented by coexpression with the KCNQ1 beta subunit, KCNE1 (minK, IsK). Northern blot analyses reveal that KCNQ2 and KCNQ3 exhibit similar expression patterns in different regions within the brain. Interestingly, coexpression of KCNQ2 and KCNQ3 results in a substantial synergistic increase in current amplitude. Coexpression of KCNE1 with the two channels strongly suppressed current amplitude and slowed kinetics of activation. The pharmacological and biophysical properties of the K+ currents observed in the coinjected oocytes differ somewhat from those observed after injecting either KCNQ2 or KCNQ3 by itself. The functional interaction between KCNQ2 and KCNQ3 provides a framework for understanding how mutations in either channel can cause a form of idiopathic generalized epilepsy.

Copper deprivation of Saccharomyces cerevisiae induces transcription of the FRE1 and CTR1 genes. FRE1 encodes a surface reductase capable of reducing and mobilizing copper chelates outside the cell, and CTR1 encodes a protein mediating copper uptake at the plasma membrane. In this paper, the protein encoded by MAC1 is identified as the factor mediating this homeostatic control. A novel dominant allele of MAC1, MAC1(up2), is mutated in a Cys-rich domain that may function in copper sensing (a G to A change of nucleotide 812 resulting in a Cys-271 to Tyr substitution). This mutant is functionally similar to the MAC1(up1) allele in which His-279 in the same domain has been replaced by Gln. Both mutations confer constitutive copper-independent expression of FRE1 and CTR1. A sequence including the palindrome TTTGCTCA ... TGAGCAAA, appearing within the 5'-flanking region of the CTR1 promoter, is necessary and sufficient for the copper- and MAC1-dependent CTR1 transcriptional regulation. An identical sequence appears as a direct repeat in the FRE1 promoter. The data indicate that the signal resulting from copper deprivation is transduced via the Cys-rich motif of MAC1 encompassing residues 264-279. MAC1 then binds directly and specifically to the CTR1 and FRE1 promoter elements, inducing transcription of those target genes. This model defines the homeostatic mechanism by which yeast regulates the cell acquisition of copper in response to copper scarcity or excess.

SHP-1 is a protein-tyrosine phosphatase associated with inhibition of activation pathways in hematopoietic cells. The catalytic activity of SHP-1 is regulated by its two SH2 (Src homology 2) domains; phosphotyrosine peptides that bind to the SH2 domains activate SHP-1. The consensus sequence (I/V)XYXX(L/V) is present in the cytoplasmic tails of several lymphocyte receptors that interact with the second SH2 domain of SHP-1. In several of these receptors, there are two or three occurrences of the motif. Here we show that the conserved hydrophobic amino acid preceding the phosphotyrosine is critical for binding to and activation of SHP-1 by peptides corresponding to sequences from killer cell inhibitory receptors. The interaction of most SH2 domains with phosphopeptides requires only the phosphotyrosine and the three residues downstream of the tyrosine. In contrast, the shortest peptide able to bind or activate SHP-1 also included the two residues upstream of the phosphotyrosine. A biphosphopeptide corresponding to the cytoplasmic tail of a killer cell inhibitory receptor with the potential to interact simultaneously with both SH2 domains of SHP-1 was the most potent activator of SHP-1. The hydrophobic residue upstream of the tyrosine was also critical in the context of the biphosphopeptide. The contribution of a hydrophobic amino acid two residues upstream of the tyrosine in the SHP-1-binding motif may be an important feature that distinguishes inhibitory receptors from those that provide activation signals.

Protein farnesyltransferase (FTase), a heterodimer enzyme consisting of alpha and beta subunits, catalyzes the addition of farnesyl groups to the C termini of proteins such as Ras. In this paper, we report that the protein substrate specificity of yeast FTase can be switched to that of a closely related enzyme, geranylgeranyltransferase type I (GGTase I) by a single amino acid change at one of the three residues: Ser-159, Tyr-362, or Tyr-366 of its beta-subunit, Dpr1. All three Dpr1 mutants can function as either FTase or GGTase I beta subunit in vivo, although some differences in efficiency were observed. These results point to the importance of two distinct regions (one at 159 and the other at 362 and 366) of Dpr1 for the recognition of the protein substrate. Analysis of the protein, after site directed mutagenesis was used to change Ser-159 to all possible amino acids, showed that either asparagine or aspartic acid at this position allowed FTase beta to function as GGTase I beta. A similar site-directed mutagenesis study on Tyr-362 showed that leucine, methionine, or isoleucine at this position also resulted in the ability of mutant FTase beta to function as GGTase I beta. Interestingly, in both position 159 and 362 substitutions, amino acids that could change the protein substrate specificity had similar van der Waals volumes. Biochemical characterization of the S159N and Y362L mutant proteins showed that their kcat/Km values for GGTase I substrate are increased about 20-fold compared with that of the wild type protein. These results demonstrate that the conversion of the protein substrate specificity of FTase to that of GGTase I can be accomplished by introducing a distinct size amino acid at either of the two residues, 159 and 362.

Several human cDNAs encoding a histone deacetylase protein, HDAC3, have been isolated. Analysis of the predicted amino acid sequence of HDAC3 revealed an open reading frame of 428 amino acids with a predicted molecular mass of 49 kDa. The HDAC3 protein is 50% identical in DNA sequence and 53% identical in protein sequence compared with the previously cloned human HDAC1. Comparison of the HDAC3 sequence with human HDAC2 also yielded similar results, with 51% identity in DNA sequence and 52% identity in protein sequence. The expressed HDAC3 protein is functionally active because it possesses histone deacetylase activity, represses transcription when tethered to a promoter, and binds transcription factor YY1. Similar to HDAC1 and HDAC2, HDAC3 is ubiquitously expressed in many different cell types.

The biochemical mechanism for the regulation of enzyme activity by lipid modulators and the role of the amphipathic alpha-helical domain of CTP:phosphocholine cytidylyltransferase (CT) was investigated by analyzing the kinetic properties of the wild-type protein and two truncation mutants isolated from a baculovirus expression system. The CT[delta 312-367] mutant protein lacked the carboxyl-terminal phosphorylation domain and retained high catalytic activity along with both positive and negative regulation by lipid modulators. The CT[delta 257-367] deletion removed in addition the region containing three consecutive amphipathic alpha-helical repeats. The CT[delta 257-367] mutant protein exhibited a significantly lower specific activity compared to CT or CT[delta 312-367] when expressed in either insect or mammalian cells; however, CT[delta 257-367] activity was refractory to either stimulation or inhibition by lipid regulators. Lipid activators accelerated CT activity by decreasing the Km for CTP from 24.7 mM in their absence to 0.7 mM in their presence. The Km for phosphocholine was not affected by lipid activators. The activity of CT[delta 257-367] was comparable to the activity of wild-type CT in the absence of lipid activators and the CTP Km for CT[delta 257-367] was 13.9 mM. The enzymatic properties of the CT[delta 231-367] mutant were comparable to those exhibited by the CT[257-367] mutant indicating that removal of residues 231 through 257 did not have any additional influence on the lipid regulation of the enzyme. Thus, the region between residues 257 and 312 was required to confer lipid regulation on CT, and the association of activating lipids with this region of the protein stimulated catalysis by increasing the affinity of the enzyme for CTP.

The phosphorylation and regulation of the URA7-encoded CTP synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)) from Saccharomyces cerevisiae by cAMP-dependent protein kinase (protein kinase A) were examined. Protein kinase A is the principal mediator of signals transmitted through the RAS/cAMP pathway in S. cerevisiae. The results of labeling experiments indicated that the phosphorylation of CTP synthetase was mediated by the RAS/cAMP pathway in vivo. In vitro, protein kinase A phosphorylated CTP synthetase at a serine residue with a stoichiometry consistent with one phosphorylation site per CTP synthetase subunit. Protein kinase A activity was dose- and time-dependent using CTP synthetase as a substrate. The dependence of protein kinase A activity on CTP synthetase was cooperative (n = 1.8) and the Km value for CTP synthetase was 73 nM. Phosphorylation of CTP synthetase with protein kinase A resulted in the stimulation (190%) of activity. The mechanism of this stimulation included an increase in the Vmax of the reaction with respect to UTP and ATP, a decrease in the Km for ATP, and a decrease in the cooperative kinetic behavior of the enzyme. Phosphorylated CTP synthetase was less sensitive to product inhibition by CTP. Protein kinase C also phosphorylates and activates CTP synthetase. Phosphorylation of CTP synthetase with protein kinases A and C together resulted in an increase in CTP synthetase activity that was slightly greater than that obtained when the enzyme was phosphorylated with either protein kinase alone.

The sequences of the metabotropic glutamate receptors (mGluRs) show little homology with other members of the G protein-coupled receptor family and exhibit several distinctive features, including a large N-terminal extracellular domain with 17 cysteines in conserved positions. Here we demonstrate that mGluR5, as well as other mGluRs, behave as species approximately twice as large as expected from their sequence, but reducing conditions cause a decrease to the predicted molecular mass. Co-immunoprecipitation experiments using wild type and epitope-tagged receptors demonstrate that this is due to specific, disulfide-dependent dimerization of the receptor. The intermolecular disulfide that mediates dimerization occurs in the extracellular domain, within about 17 kDa from the N terminus.

Cdc42 plays an important role in intracellular signaling pathways that influence cell morphology and motility and stimulate DNA synthesis. In attempts to determine whether nonreceptor tyrosine kinases play a fundamental role in Cdc42 signaling, we have cloned and biochemically characterized a new Cdc42-associated tyrosine kinase (ACK) from bovine brain. This tyrosine kinase, named ACK-2, has a calculated molecular mass of 83 kDa and shares a number of primary structural domains with the 120-kDa ACK (ACK-1). The main differences between the primary structures of ACK-2 and ACK-1 occur in the amino- and carboxyl-terminal regions. Like ACK-1, ACK-2 binds exclusively to activated (GTP-bound) Cdc42 and does not bind to its closest homologs, e.g. activated Rac. ACK-2 could not be activated by addition of glutathione S-transferase (GST)-Cdc42(Q61L), a GTPase-defective mutant, or by GTPgammaS-loaded GST-Cdc42 in in vitro kinase assays. However, ACK-2 was activated when cotransfected with wild type Cdc42 or Cdc42(Q61L) and stably associated with Cdc42(Q61L) in vivo, indicating that ACK-2 interacts with active Cdc42 in cells. Furthermore, the tyrosine kinase activity of ACK-2 was stimulated both by epidermal growth factor and bradykinin, suggesting that ACK-2 may play a role in the signaling actions of both receptor tyrosine kinases or heterotrimeric G-protein-coupled receptors.

The role of the phosphorylated carboxyl-terminal domain of CTP:phosphocholine cytidylyltransferase (CT) in the regulation of enzyme activity was investigated by comparing the catalytic properties of wild-type CT to two mutant proteins with altered carboxyl-terminal phosphorylation domains. CT isolated from a baculovirus expression system was extensively phosphorylated at multiple sites in the carboxyl-terminal domain. The CT[S315A] mutant lacked a major CT phosphorylation site, and the carboxyl-terminal deletion mutant, CT[delta 312-367], was not phosphorylated. The higher activities of CT[delta 312-367] and CT[S315A] relative to CT were attributed to differences in the sensitivities of the enzymes to lipid activators. The rank order of the apparent Km values for activation by either phosphatidylcholine/oleic acid or phosphatidylcholine/diacylglycerol was CT > CT[S315A] > CT[delta 312-367]. In addition, CT exhibited negative cooperativity in its activation by phosphatidylcholine/oleic acid (nH = 0.64) and phosphatidylcholine/diacylglycerol (nH = 0.74) vesicles, whereas CT[delta 312-367] and CT[S315A] did not. These data support the concept that the phosphorylation of the CT carboxyl-terminal domain interferes with the activation of CT by lipid regulators.

The URA7- and URA8-encoded CTP synthetases (EC 6.3.4.2, UTP:ammonia ligase (ADP-forming) are functionally overlapping enzymes responsible for the biosynthesis of CTP in the yeast Saccharomyces cerevisiae. URA8-encoded CTP synthetase was purified to apparent homogeneity by ammonium sulfate fractionation of the cytosolic fraction followed by chromatography with Q-Sepharose, Affi-Gel Blue, Mono Q, and Superose 6. The subunit molecular mass (67 kDa) of purified URA8-encoded CTP synthetase was in good agreement with the predicted size of the URA8 gene product. Antibodies raised against a fusion protein constructed from the coding sequences of the URA8 gene and expressed in Escherichia coli reacted with purified URA8-encoded CTP synthetase. Native URA8-encoded CTP synthetase existed as a dimer which oligomerized to a tetramer in the presence of its substrates UTP and ATP. Maximum URA8-encoded CTP synthetase activity was dependent on Mg2+ ions (Ka = 2.4 mM) and 2-mercaptoethanol at the pH optimum of 7.5. The enzyme followed saturation kinetics toward UTP (Km = 74 microM), ATP (Km = 22 microM), and glutamine (Km = 0.14 mM). GTP stimulated (Ka = 26 microM) URA8-encoded CTP synthetase activity 12-fold. CTP potently inhibited (IC50 = 85 microM) URA8-encoded CTP synthetase activity and, in addition, caused the dependence of activity toward UTP to become cooperative. The URA8-encoded CTP synthetase and the previously purified URA7-encoded CTP synthetase differed significantly with respect to several biochemical properties including turnover number, pH optimum, substrate dependences, and sensitivity to inhibition by CTP. The URA7-encoded CTP synthetase mRNA was 2-fold more abundant when compared with URA8-encoded CTP synthetase mRNA. Both CTP synthetase isoforms were maximally expressed in the exponential phase of growth.

Phosphorylation of CTP synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)) from Saccharomyces cerevisiae protein kinase C was examined. Using pure CTP by synthetase as a substrate, protein kinase C activity was dose- and time-dependent and required calcium, diacylglycerol, and phosphatidylserine for full activation. Protein kinase C activity was also dependent on the concentration of CTP synthetase. Protein kinase C phosphorylated CTP synthetase on serine and threonine residues in vitro whereas the enzyme was primarily phosphorylated on serine residues in vivo. Phosphopeptide mapping analysis of CTP synthetase phosphorylated in vitro and in vivo indicated that the enzyme was phosphorylated on more than one site. Most of the phosphopeptides derived from CTP synthetase phosphorylated in vivo were the same as those derived from CTP synthetase phosphorylated by protein kinase C in vitro. The stoichiometry of the phosphorylation of native CTP synthetase was 0.4 mol of phosphate/mol of enzyme whereas the stoichiometry of the phosphorylation of alkaline phosphatase-treated CTP synthetase was 2.2 mol of phosphate/mol of enzyme. This indicated that CTP synthetase was purified in a phosphorylated state. Phosphorylation of CTP synthetase resulted in a 3-fold activation in enzyme activity whereas alkaline phosphatase treatment of CTP synthetase resulted in a 5-fold decrease in enzyme activity. Overall, the results reported here were consistent with the conclusion that CTP synthetase was regulated by protein kinase C phosphorylation.

YY1 is a zinc finger transcription factor with unusual structural and functional features. In a yeast two-hybrid screen, two cellular proteins, cyclophilin A (CyPA) and FK506-binding protein 12 (FKBP12), interacted with YY1. These interactions are specific and also occur in mammalian cells. Cyclosporin A and FK506 efficiently disrupt the YY1-CyPA and YY1-FKBP12 interactions. Overexpression of human CyPA and FKBP12 have different effects on YY1-regulated transcription with these effects being promoter-dependent. These results suggest that immunophilins may be mediators in the functional role of YY1.

A unique protein with an apparent molecular mass of 96 kilodaltons (p96) was detected in the murine macrophage cell line, BAC1.2F5. The murine cDNA encoding p96 was cloned and sequenced, along with cDNAs representing two alternatively spliced forms of the protein. All three proteins possessed identical amino-terminal domains with significant similarity to the amino-terminal domain of the Drosophila disabled gene product and carboxyl-terminal domains containing proline-rich sequences characteristic of src homology region (domain 3) binding regions. BAC1.2F5 cells predominantly expressed the p96 protein, although mRNA and protein corresponding to the p67 splice variant were also detected. Electrophoretic gel retardation of p96 in response to stimulation of the cells with colony-stimulating factor 1 was noticeable within 5 min after growth factor addition and reached a maximum at 60 min. Metabolic labeling experiments showed that the gel retardation of p96 was associated with increased phosphorylation of the protein exclusively on serine residues. These data identify a novel protein that is phosphorylated in response to mitogenic growth factor stimulation.

Histone deacetylase-2 (HDAC2) is a component of a complex that mediates transcriptional repression in mammalian cells. A mouse HDAC2 cDNA was used to identify several recombinant clones containing the entire mouse HDAC2 gene. The mouse HDAC2 gene spans over 36 kilobase pairs and is composed of 14 exons (ranging from 58 to 362 nucleotides in length) and 13 introns (ranging from 75 base pairs to 19 kilobase pairs in length). Primer extension analysis with total RNA from NIH3T3 cells revealed a major transcriptional start site at 221 base pairs 5' of the ATG translational start codon. Upstream of the transcriptional start site, no canonical TATA box was found, but binding sites for several known transcription factors were identified. Transient transfection studies with 5' deletion mutants localized the promoter to no more than 76 base pairs upstream from the major transcriptional start site. Fluorescence in situ hybridization mapped mouse HDAC2 to chromosomal location 10B1, which is in close proximity to the growth factor-inducible gene fisp-12. Information concerning the genomic organization and promoter of HDAC2 will be useful in studies of the regulation of histone deacetylase activities, which in turn are important in studies of the regulation of transcriptional repression in mammalian cells.