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S Baksh

Publications and source records attributed to S Baksh.

12 recordsLinked to original sources
PubMed2000

Cutting edge: gab2 mediates an inhibitory phosphatidylinositol 3'-kinase pathway in T cell antigen receptor signaling.

Phosphatidylinositol 3'-kinase (PI3K) is a key component of multiple signaling pathways, where it typically promotes survival, proliferation, and/or adhesion. Here, we show that in TCR signaling, the scaffolding adapter Gab2 delivers an inhibitory signal via PI3K. Overexpression of Gab2 in T cell lines inhibits TCR-evoked activation of the IL-2 promoter, blocking NF-AT- and NF-kappaB-directed transcription. Inhibition is abrogated by mutating the Gab2 p85-binding sites, by treatment with PI3K inhibitors or by cotransfection of phosphatase homolog of tensin. Our findings provide the first evidence of a negative function for a scaffolding adapter in T cells and identify Gab2/PI3K-containing complexes as novel regulators of TCR signaling.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Calcineurin regulation of the mammalian G0/G1 checkpoint element, cyclin dependent kinase 4.

Cyclin dependent kinase 4 (cdk4) activity is controlled by the binding of regulatory subunits and inhibitory factors, as well as tyrosine and serine/threonine phosphorylation. More recently the influence of calcium levels have been demonstrated. Using transient transfections in Jurkat cells, we observed specific binding between cdk4 and the calcium and calmodulin activated serine/threonine phosphatase, calcineurin. Furthermore, we demonstrated that the inhibition of the phosphatase activity of calcineurin with FK506 and cyclosporin A resulted in an overall increase in cdk4 kinase activity, suggesting that the phosphatase activity of calcineurin was inhibitory to the kinase activity of cdk4. In contrast, we were not able to observe a similar effect on the kinase activity of either cdk6 or cdk2, indicating that the phosphatase activity of calcineurin was specific for cdk4. In addition, using an in vitro phosphatase assay for calcineurin, we observed that the exogenous addition of calcineurin resulted in the dephosphorylation of cdk4, an event that downregulated the kinase activity of cdk4. Calcineurin could, therefore, play an opposing role to the action of the cyclin activating kinase complex, an enzyme that upregulates the kinase activity of cdk4, an important G0/G1 checkpoint element in mammalian cells. Oncogene (2000) 19, 2820 - 2827

Calcineurin↗
PubMed2000

The role of calcineurin in lymphocyte activation.

Lymphokine gene transcription involves numerous signal transduction molecules and second messengers. The serine/threonine phosphatase calcineurin has been demonstrated to play a central role in the immediate, early activation of numerous lymphokines (such as interleukin [IL]-2) and in the regulation of cell surface receptors such as CD40L, CD95, and recently CD25 alpha (the alpha chain of the IL-2 receptor). In addition to lymphocyte activation, calcineurin functions include control of neuronal signaling, muscle contraction, muscle hypertrophy and cellular death. Therefore, calcineurin not only plays a vital role in the regulation of T lymphocyte function, but also functions in cellular environments outside the immune system.

Animals↗
PubMed1995

Interaction of calreticulin with protein disulfide isomerase.

We report here that calreticulin interacts with protein disulfide isomerase (PDI). The PDI-calreticulin complex can be dissociated by Zn(2+)-iminodiacetate-substituted Sepharose-agarose chromatography, suggesting that these interactions may be Zn2+-dependent. Direct interaction between calreticulin and PDI is also documented by calreticulin affinity chromatography. PDI was the only pancreatic microsomal protein retained on the calreticulum affinity column. Calreticulin and PDI were identified by their NH2-terminal amino acid sequence analysis, mobilities in SDS-polyacrylamide gel electrophoresis, binding of 45Ca2+, and their reactivity with specific antibodies. Using glutathione S-transferase-calreticulin fusion proteins, we show that PDI interacts strongly with the P-domain and only weakly with the N-domain of calreticulin. Expression of calreticulin domains and PDI as fusion proteins with GAL4 in the yeast two-hybrid system revealed that calreticulin interacted with PDI also under normal cellular conditions. Interaction with PDI required only the NH2-terminal region of the N-domain (amino acid residues 1-83) and the P-domain (amino acid residues 150-240) of calreticulin. Importantly, interaction between calreticulin and PDI led to the modulation of their activities. In the presence of PDI, calreticulin does not bind Ca2+ with high affinity. Calreticulin or the N-domain of calreticulin inhibited PDI ability to refold scrambled RNase A.

Amino Acid Sequence↗
PubMed1995

Identification of the Zn2+ binding region in calreticulin.

Calreticulin binds Zn2+ with the relatively high affinity/low capacity. To determine the location of the Zn2+ binding site in calreticulin different domains of the protein were expressed in E. coli, using the glutathione S-transferase fusion protein system, and their Zn(2+)-dependent interaction with Zn(2+)-IDA-agarose were determined. Three distinct domains were used in this study: the N + P-domain (the first 290 residues); the N-domain (residues 1-182) and the proline-rich P-domain (residues 180-273). The N + P-domain bound to the Zn(2+)-IDA-agarose and were eluted with an increasing concentration of imidazole. The N-domain also bound 65Zn2+ as measured by the overlay method. The P-domain did not interact with the Zn(2+)-IDA-agarose and it did not bind any detectable amount of Zn2+. Chemical modification of calreticulin with diethyl pyrocarbonate indicated that five out of seven histidines were protected in the presence of Zn2+ but they were modified by diethyl pyrocarbonate in the absence of Zn2+ suggesting that these residues may be involved in Zn2+ binding to calreticulin. We conclude that Zn2+ binding sites in calreticulin are localized to the N-domain of the protein, region that is not involved in Ca2+ binding to calreticulin.

Binding Sites↗
PubMed1995

Zn2+ binding to cardiac calsequestrin.

Zn2+ binding to canine cardiac calsequestrin was investigated using the Zn2+ specific fluorescence dye salicylcarbohydrazone (SACH), 65Zn2+ overlay and Zn(2+)-IDA chromatography. Cardiac calsequestrin binds approximately 200 moles of Zn2+/mole of protein with the Kd = 300 microM. Zn2+ binding to calsequestrin was further confirmed by 65Zn2+ overlay and Zn(2+)-dependent aggregation of the protein. However, calsequestrin did not bind to a Zn(2+)-IDA-agarose column, indicating that histidine residues may not be involved in Zn2+ binding to the protein. Circular dichroism revealed only minor Zn(2+)-dependent conformational changes in calsequestrin. We conclude that calsequestrin is a Ca(2+)- and Zn(2+)-binding protein and that Zn2+ may modulate the structure and function of the protein.

Animals↗
PubMed1994

Crystallization of canine cardiac calsequestrin.

Calsequestrin is the major Ca2+ binding protein in the lumen of the sarcoplasmic reticulum membranes. Two X-ray quality crystal forms of canine cardiac calsequestrin were obtained by the hanging drop method using KCl as a precipitant. One form is monoclinic (space group P2(1), a = 73.4 A, b = 104.4 A, c = 60.2 A, beta = 120.4 degrees) with two molecules in the asymmetric unit and a solvent content of approximately 40%. The second form is trigonal (P3(1)21 or P3(2)21, a = b = 99.3 A, c = 89.8 A) with a single molecule in the asymmetric unit and 55% solvent content. Cross rotation function calculations show that despite the different space groups the packing of the molecules in both crystals is likely to be similar suggesting the existence of a stable dimer. The monoclinic crystals diffract beyond 3 A using a laboratory rotating anode source, while under the same conditions the trigonal crystals diffract only to approximately 4.5 A. This is the first report of successful preparation of X-ray quality crystals of a high capacity Ca2+ binding protein.

Animals↗
PubMed1992

Expression and purification of recombinant and native calreticulin.

Calreticulin is a 60-kDa Ca(2+)-binding protein of the endo(sarco)plasmic reticulum membranes of a variety of cellular systems. The protein binds approximately 25 mol of Ca2+ with low affinity and approximately 1 mol of Ca2+ with high affinity and is believed to be a site for Ca2+ binding/storage in the lumen of the endo(sarco)plasmic reticulum. In the present study, we describe purification procedures for the isolation of recombinant and native calreticulin. Recombinant calreticulin was expressed in Escherichia coli, using the glutathione S-transferase fusion protein system, and was purified to homogeneity on glutathione-Sepharose followed by Mono Q FPLC chromatography. A selective ammonium sulfate precipitation method was developed for the purification of native calreticulin. The protein was purified from ammonium sulfate precipitates by diethylaminoethyl-Sephadex and hydroxylapatite chromatography procedures, which eliminates the need to prepare membrane fractions. The purification procedures reported here for recombinant and native calreticulin yield homogeneous preparations of the proteins, as judged by the HPLC reverse-phase chromatography and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified native and recombinant calreticulin were identified by their NH2-terminal amino acid sequences, by their Ca2+ binding properties, and by their reactivity with anticalreticulin antibodies.

Amino Acid Sequence↗
PubMed1991

Expression of calreticulin in Escherichia coli and identification of its Ca2+ binding domains.

Recombinant calreticulin and discrete domains of calreticulin were expressed in Escherichia coli, using the glutathione S-transferase fusion protein system, and their Ca2+ binding properties were determined. Native calreticulin bound 1 mol of Ca2+/mol of protein with high affinity, and also bound approximately 20 mol of Ca2+/mol of protein with low affinity. Both Ca2+ binding sites were present in the recombinant calreticulin indicating that proper folding of the protein was achieved using this system. Calreticulin is structurally divided into three distinct domains: the N-domain encompassing the first 200 residues; the P-domain which is enriched in proline residues (residue 187-317); and the C-domain which covers the carboxyl-terminal quarter of the protein (residues 310-401), and contains a high concentration of acidic residues. These domains were expressed in E. coli, isolated, and purified, and their Ca2+ binding properties were analyzed. The C-domain bound approximately 18 mol of Ca2+/mol of protein with a dissociation constant of approximately 2 mM. The P-domain bound approximately 0.6-1 mol of Ca2+/mol of protein with a dissociation constant of approximately 10 microM. The P-domain and the C-domain, when expressed together as the P+C-domain, bound Ca2+ with both high affinity and low affinity, reminiscent of both full length recombinant calreticulin and native calreticulin. In contrast the N-domain, did not bind any detectable amount of 45Ca2+. We conclude that calreticulin has two quite distinct types of Ca2+ binding sites, and that these sites are in different structural regions of the molecule. The P-domain binds Ca2+ with high affinity and low capacity, whereas the C-domain binds Ca2+ with low affinity and high capacity.

Amino Acid Sequence↗
PubMed1991

Calreticulin, and not calsequestrin, is the major calcium binding protein of smooth muscle sarcoplasmic reticulum and liver endoplasmic reticulum.

The distribution of calsequestrin and calreticulin in smooth muscle and non-muscle tissues was investigated. Immunoblots of endoplasmic reticulum proteins probed with anti-calreticulin and anti-calsequestrin antibodies revealed that only calreticulin is present in the rat liver endoplasmic reticulum. Membrane fractions isolated from uterine smooth muscle, which are enriched in sarcoplasmic reticulum, contain a protein band which is immunoreactive with anti-calreticulin but not with anti-calsequestrin antibodies. The presence of calreticulin in these membrane fractions was further confirmed by 45Ca2+ overlay and "Stains-All" techniques. Calreticulin was also localized to smooth muscle sarcoplasmic reticulum by the indirect immunofluorescence staining of smooth muscle cells with anti-calreticulin antibodies. Furthermore, both liver and uterine smooth muscle were found to contain high levels of mRNA encoding calreticulin, whereas no mRNA encoding calsequestrin was detected. We have employed an ammonium sulfate precipitation followed by Mono Q fast protein liquid chromatography, as a method by which calsequestrin and calreticulin can be isolated from whole tissue homogenates, and by which they can be clearly resolved from one another, even where present in the same tissue. Calreticulin was isolated from rabbit and bovine liver, rabbit brain, rabbit and porcine uterus, and bovine pancreas and was identified by its amino-terminal amino acid sequence. Calsequestrin cannot be detected in preparations from whole liver tissue, and only very small amounts of calsequestrin are detectable in ammonium sulfate extracts of uterine smooth muscle. We conclude that calreticulin, and not calsequestrin, is a major Ca2+ binding protein in liver endoplasmic reticulum and in uterine smooth muscle sarcoplasmic reticulum. Calsequestrin and calreticulin may perform parallel functions in the lumen of the sarcoplasmic and endoplasmic reticulum.

Amino Acid Sequence↗
PubMed1991

Identification and immunolocalization of calreticulin in pancreatic cells: no evidence for "calciosomes".

In the present study, we have shown that calreticulin is a major Ca(2+)-sequestering protein in pancreatic microsomes. This protein is a peripheral membrane protein and could be extracted from the microsomal membrane with carbonate buffer at pH 11.4. Calreticulin was identified in the membrane fractions by immunoblotting with a specific antibody, by a 45Ca2+ overlay technique, and by NH2-terminal amino acid analysis of the purified protein. Immunocytochemical localization of calreticulin in pancreatic acinar cells and pancreatic fibroblasts showed that the protein is localized to the ER membranes in these cells. We were unable to detect calsequestrin or any calsequestrin-like proteins in the pancreas and found no evidence for the existence of large numbers of specialized, calreticulin-containing vesicles which could be an equivalent of the calsequestrin-containing calciosomes previously reported in this tissue. Purified pancreatic calreticulin binds Ca2+ with both a low and a high capacity (approximately 1 mol of Ca2+/mol of protein and approximately 20-23 mol of Ca2+/mol of protein). The concentrations of Ca2+ required for half-maximal saturation of the low and high capacity sites were approximately 4-6 microM and approximately 1.5 mM, respectively. We conclude that calreticulin, which is confined to the lumen of the ER, plays a major role in Ca2+ storage in pancreatic cells.

Amino Acid Sequence↗