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Biomedical subjects

S Pestka

Publications and source records attributed to S Pestka.

At least 37 records · Page 2Linked to original sources
PubMed1999

The human homologue of the yeast proteins Skb1 and Hsl7p interacts with Jak kinases and contains protein methyltransferase activity.

To expand our understanding of the role of Jak2 in cellular signaling, we used the yeast two-hybrid system to identify Jak2-interacting proteins. One of the clones identified represents a human homologue of the Schizosaccaromyces pombe Shk1 kinase-binding protein 1, Skb1, and the protein encoded by the Saccharomyces cerevisiae HSL7 (histone synthetic lethal 7) gene. Since no functional motifs or biochemical activities for this protein or its homologues had been reported, we sought to determine a biochemical function for this human protein. We demonstrate that this protein is a protein methyltransferase. This protein, designated JBP1 (Jak-binding protein 1), and its homologues contain motifs conserved among protein methyltransferases. JBP1 can be cross-linked to radiolabeled S-adenosylmethionine (AdoMet) and methylates histones (H2A and H4) and myelin basic protein. Mutants containing substitutions within a conserved region likely to be involved in AdoMet binding exhibit little or no activity. We mapped the JBP1 gene to chromosome 14q11.2-21. In addition, JBP1 co-immunoprecipitates with several other proteins, which serve as methyl group acceptors and which may represent physiological targets of this methyltransferase. Messenger RNA for JBP1 is widely expressed in human tissues. We have also identified and sequenced a homologue of JBP1 in Drosophila melanogaster. This report provides a clue to the biochemical function for this conserved protein and suggests that protein methyltransferases may have a role in cellular signaling.

Amino Acid Sequence↗
PubMed1999

The intracellular domain of interferon-alpha receptor 2c (IFN-alphaR2c) chain is responsible for Stat activation.

Type I IFNs activate the Jak-Stat signal transduction pathway. The IFN-alpha receptor 1 (IFN-alphaR1) subunit and two splice variants of the IFN-alphaR2 subunit, IFN-alphaR2c and IFN-alphaR2b, are involved in ligand binding. All these receptors have been implicated in cytokine signaling and, specifically, in Stat recruitment. To evaluate the specific contribution of each receptor subunit to Stat recruitment we employed chimeric receptors with the extracellular domain of either IFN-gammaR2 or IFN-gammaR1 fused to the intracellular domains of IFN-alphaR1, IFN-alphaR2b, and IFN-alphaR2c. These chimeric receptors were expressed in hamster cells. Because human IFN-gamma exhibits no activity on hamster cells, the use of the human IFN-gamma receptor extracellular domains allowed us to avoid the variable cross-species activity of the type I IFNs and eliminate the possibility of contributions of endogenous type I IFN receptors into the Stat recruitment process. We demonstrate that Stat recruitment is solely a function of the IFN-alphaR2c intracellular domain. When chimeric receptors with the human IFN-gammaR1 extracellular domain and various human IFN-alpha receptor intracellular domains were expressed in hamster cells carrying the human IFN-gammaR2 subunit, only the IFN-alphaR2c subunit was capable of supporting IFN-gamma signaling as measured by MHC class I induction, antiviral protection, and Stat activation. Neither the IFN-alphaR2b nor the IFN-alphaR1 intracellular domain was able to recruit Stats or support IFN-gamma-induced biological activities. Thus, the IFN-alphaR2c intracellular domain is necessary and sufficient to activate Stat1, Stat2, and Stat3 proteins.

Animals↗
PubMed1999

Construction of phosphorylatable chimeric monoclonal antibody CC49 with a casein kinase I recognition site.

Phosphorylation sites for casein kinase I were introduced into chimeric monoclonal antibody CC49 (MAb-chCC49) by inserting a synthetic fragment (CK1) encoding two casein kinase I phosphorylation sites into an expression vector. The phosphorylation sites were created by incorporating the predicted consensus sequences for phosphorylation by the casein kinase I at the carboxyl terminus of the heavy-chain constant region of the MAb-chCC49. The resultant modified MAb-chCC49 (MAb-chCC49CK1) was expressed and purified. The MAb-chCC49CK1 protein can be phosphorylated by the casein kinase I with [gamma-32P]ATP to high radiospecific activity. The 32P-labeled MAb-chCC49CK1 protein binds to cells expressing TAG-72 antigens. The introduction of phosphorylation sites into MAb provides new reagents for the diagnosis and treatment of cancer. This demonstrates that, as was described for the cAMP-dependent protein kinase site, the casein kinase I recognition site can also be used to introduce phosphorylation sites into proteins.

Amino Acid Sequence↗
PubMed1999

Introduction of protein kinase recognition sites into proteins: a review of their preparation, advantages, and applications.

Labeled proteins are used in a variety of applications. This review focuses on methods that utilize genetic engineering to introduce protein kinase recognition sites into proteins. Many protein kinase recognition sites can be introduced into proteins and serve as useful tags for a variety of purposes. The introduction of protein kinase recognition sites into proteins can be achieved without modifying the essential structure or function of the proteins. Because proteins modified by these procedures retain their activity after phosphorylation, they can be used in many applications. The phosphorylatable proteins can be labeled easily to high specific activity with radioisotopes ((32)P, (33)P, or (35)S), or the nonradioactive (31)P can be used. The use of these radioisotopes provides a convenient and safe method for radiolabeling proteins. Moreover, the use of the nonradioactive (31)P with protein tyrosine kinase recognition sites permits the tagging of proteins and their detection with the many anti-phosphotyrosine antibodies available. Overall, the procedure represents a convenient, safe, and efficient method to label proteins for a variety of applications.

Amino Acid Motifs↗
PubMed1999

Antigenic characterization of recombinant, lymphoblastoid, and leukocyte IFN-alpha by monoclonal antibodies.

To gain more insight into similarities of different interferon-alpha (IFN-alpha) species, we evaluated neutralization and immunoactivity of a variety of IFN preparations with various monoclonal antibodies (IFN-alpha mAb). Nine IFN-alpha mAb obtained through immunization with recombinant IFN-alpha (rmAb), lymphoblastoid IFN-alpha (LY mAb), and leukocyte IFN-alpha (LE mAb) were tested. The IFN-alpha mAb were evaluated for their ability to neutralize the antiviral activity of 11 recombinant IFN-alpha subtypes, two recombinant IFN-alpha hybrids, and lymphoblastoid and leukocyte IFN-alpha preparations. The same IFN-alpha mAb were also used in immunoblotting, and some of them were used in immunoaffinity chromatography. The results of the neutralization assay reveal that the IFN-alpha mAb significantly differ in their ability to neutralize the individual IFN-alpha species. Interestingly, none of the IFN-alpha mAb was able to neutralize all the IFN-alpha species. In particular, rmAb were unable to neutralize LE-IFN-alpha or LY-IFN-alpha, whereas LE mAb and LY mAb efficiently neutralized rIFN-alpha2. In some cases, the epitopes to which IFN-alpha mAb are directed were identified through the use of synthetic fragments of IFN-alpha2 or by evaluating the selectivity in binding to IFN-alpha subtypes.

Animals↗
PubMed1998

Targeted disruption of the interferon-gamma receptor 2 gene results in severe immune defects in mice.

To study the role of the interferon- (IFN) gammaR2 chain in IFN-gamma signaling and immune function, IFN-gammaR2-deficient mice have been generated and characterized. Cells derived from IFN-gammaR2 -/- mice are unable to activate either JAK/STAT signaling proteins or gene transcription in response to IFN-gamma. The lack of IFN-gamma responsiveness alters IFN-gamma-induced Ig class switching by B cells from these mice. In vitro cultures of T cells demonstrate that the T cells from the IFN-gammaR2 -/- mice have a defect in Th1 cell differentiation. The IFN-gammaR2 (-/-) mice also produce lower amounts of IFN-gamma in response to antigenic challenge. In addition, IFN-gammaR2 -/- mice are defective in contact hypersensitivity and are highly susceptible to infection by Listeria monocytogenes. These results demonstrate that the IFN-gammaR2 is essential for IFN-gamma-mediated immune responses in vivo.

Animals↗
PubMed1998

Biological properties of recombinant alpha-interferons: 40th anniversary of the discovery of interferons.

IFNs were first described as potent antiviral agents 40 years ago, and recombinant IFN-alpha2a and IFN-alpha2b were approved for the treatment of hairy cell leukemia just 11 years ago. Today, alpha-IFNs are approved worldwide for the treatment of a variety of malignancies and virologic diseases. Although the exact mechanism of action of IFN-alpha in the treatment of such diseases is not fully understood, many advances have been made in the characterization of the physicochemical and diverse biological properties of this highly pleiotropic cytokine. Here we review recent developments in our understanding of the antiviral and immunoregulatory properties of IFN-alpha, the nature of the multisubunit IFN-alpha receptor, and the molecular mechanisms of signal transduction. Where available, we have included comparative data on recombinant alpha-IFNs derived from both naturally occurring and nonnaturally occurring synthetic genes. We also review clinical data and data on the side effects and antigenicity of different sources of recombinant alpha-IFNs in humans. These latter topics are of clinical interest, because they may potentially affect the efficacy of these various products. Hopefully, what is already known about IFN will prompt further exploration into the mechanism(s) of action of IFN-alpha and thus deliver new applications for this prototypic cytokine, whose full therapeutic potential is yet to be realized.

Antineoplastic Agents↗
PubMed1998

Tumor cell responses to IFNgamma affect tumorigenicity and response to IL-12 therapy and antiangiogenesis.

Expression of a dominant negative mutant IFNgammaR1 in murine SCK and K1735 tumor cells rendered them relatively unresponsive to IFNgamma in vitro and more tumorigenic and less responsive to IL-12 therapy in vivo. IL-12 induced histologic evidence of ischemic damage only in IFNgamma-responsive tumors, and in vivo Matrigel vascularization assays revealed that while IFNgamma-responsive and -unresponsive tumor cells induced angiogenesis equally well, IL-12 and its downstream mediator IFNgamma only inhibited angiogenesis induced by the responsive cells. IL-12 induced angiogenesis inhibitory activity in the responsive cells, which may be attributable to production of the chemokine IP-10. Thus, IL-12 and IFNgamma inhibit tumor growth by inducing tumor cells to generate antiangiogenic activity.

Animals↗
PubMed1998

An antagonist peptide-EPO receptor complex suggests that receptor dimerization is not sufficient for activation.

Dimerization of the erythropoietin (EPO) receptor (EPOR), in the presence of either natural (EPO) or synthetic (EPO-mimetic peptides, EMPs) ligands is the principal extracellular event that leads to receptor activation. The crystal structure of the extracellular domain of EPOR bound to an inactive (antagonist) peptide at 2.7 A resolution has unexpectedly revealed that dimerization still occurs, but the orientation between receptor molecules is altered relative to active (agonist) peptide complexes. Comparison of the biological properties of agonist and antagonist EMPs with EPO suggests that the extracellular domain orientation is tightly coupled to the cytoplasmic signaling events and, hence, provides valuable new insights into the design of synthetic ligands for EPOR and other cytokine receptors.

Amino Acid Sequence↗
PubMed1998

Review of recent developments in the molecular characterization of recombinant alfa interferons on the 40th anniversary of the discovery of interferon.

Recombinant alfa interferons (IFN-alpha s) are approved worldwide for the treatment of a variety of cancers and diseases of virologic origin. A series of recent advances in the molecular characterization of recombinant IFN-alpha s have allowed the determination of the three-dimensional IFN-alpha 2b structure by high-resolution x-ray crystallography. We review here recent developments in our understanding of the molecular and physicochemical properties of recombinant IFN-alpha, including our current state of knowledge of the IFN-alpha gene family and the multiple species of human leukocyte IFN. Based on the reported three-dimensional structure of IFN-alpha 2b, we propose a molecular model for the IFN-alpha 2b receptor complex and predict models for the naturally occurring subtypes IFN-alpha 1 and IFN-alpha 8, as well as the synthetic, non-naturally occurring consensus IFN. Such models provide molecular insights into the mechanism of action of IFN-alpha.

Humans↗
PubMed1998

Construction of phosphorylatable chimeric monoclonal antibody CC49.

Phosphorylation sites were introduced into chimeric monoclonal antibody CC49 (MAb-chCC49) by inserting synthetic fragments encoding two and six phosphorylation sites into an expression vector, pdHL7. The phosphorylation sites were created by using the predicted consensus sequences for phosphorylation by the cAMP-dependent protein kinase to the carboxyl terminus of the heavy chain constant region of the MAb-chCC49. The resultant modified antibodies (MAb-chCC49K1 and MAb-chCC49-6P) were expressed in NS0 cells and purified. The MAb-chCC49K1 protein contains two phosphorylation sites per heavy chain whereas the MAb-chCC49-6P protein contains six sites per heavy chain. Both MAb-chCC49K1 and MAb-chCC49-6P proteins can be phosphorylated by the catalytic subunit of cAMP-dependent protein kinase with [gamma-32P]ATP to high specific activity. The 32P-labeled MAb-chCC49K1 and MAb-chCC49-6P proteins bind to cells expressing TAG-72 antigens. The introduction of phosphorylation sites into a monoclonal antibody provides a reagent for the diagnosis and treatment of cancer. The use of multiple phosphorylation sites provides antibodies with very high specific radioactivity and demonstrates that cassettes of phosphorylation sites can be introduced into proteins without altering their functional activity.

Amino Acid Sequence↗
PubMed1998

Construction of phosphorylatable chimeric monoclonal antibody CC49 with a tyrosine srC kinase recognition site.

A phosphorylation site for a tyrosine kinase was introduced into chimeric monoclonal antibody CC49 (MAb-chCC49) by inserting a synthetic fragment (Tyr) encoding one tyrosine kinase phosphorylation site into an expression vector. The phosphorylation site was created by incorporating the predicted consensus sequences for phosphorylation by the tyrosine kinase at the carboxyl terminus of the heavy chain constant region of the MAb-chCC49. The resultant modified MAb-chCC49 (MAb-chCC49Tyr) was expressed and purified. The MAb-chCC49Tyr protein can be phosphorylated by the tyrosine Src kinase with [gamma-32P]ATP to high radiospecific activity. The 32P-labeled MAb-chCC49Tyr protein binds to cells expressing TAG-72 antigens. The introduction of phosphorylation sites into monoclonal antibodies (MAb) provides a new reagent for the diagnosis and treatment of cancer. This demonstrates that, as was described for the cAMP-dependent protein kinase site, a tyrosine phosphorylation site can also be used to introduce phosphorylation sites into proteins.

Amino Acid Sequence↗
PubMed1997

Identification and functional characterization of a second chain of the interleukin-10 receptor complex.

Interleukin-10 (IL-10) is a pleiotropic cytokine which signals through a specific cell surface receptor complex. Only one chain, that for ligand binding (IL-10Ralpha or IL-10R1), was identified previously. We report here that, although human IL-10 binds to the human IL-10R1 chain expressed in hamster cells, it does not induce signal transduction. However, the co-expression of CRFB4, a transmembrane protein of previously unknown function belonging to the class II cytokine receptor family, together with the IL-10R1 chain renders hamster cells sensitive to IL-10. The IL-10:CRFB4 complex was detected by cross-linking to labeled IL-10. In addition, the IL-10R1 chain was able to be co-immunoprecipitated with anti-CRF antibody when peripheral blood mononuclear cells were treated with IL-10. These results demonstrate that the CRFB4 chain is part of the IL-10 receptor signaling complex. Thus, the CRFB4 chain, which we designate as the IL-10R2 or IL-10Rbeta chain, serves as an accessory chain essential for the active IL-10 receptor complex and to initiate IL-10-induced signal transduction events.

Animals↗
PubMed1997

Chimeric erythropoietin-interferon gamma receptors reveal differences in functional architecture of intracellular domains for signal transduction.

Binding of interferon gamma (IFN-gamma) causes oligomerization of the two interferon gamma receptor (IFN-gammaR) subunits, receptor chain 1 (IFN-gammaR1, the ligand-binding chain) and the second chain of the receptor (IFN-gammaR2), and causes activation of two Jak kinases (Jak1 and Jak2). In contrast, the erythropoietin receptor (EpoR) requires only one receptor chain and one Jak kinase (Jak2). Chimeras between the EpoR and the IFN-gammaR1 and IFN-gammaR2 chains demonstrate that the architecture of the EpoR and the IFN-gammaR complexes differ significantly. Although IFN-gammaR1 alone cannot initiate signal transduction, the chimera EpoR/gammaR1 (extracellular/intracellular) generates slight responses characteristic of IFN-gamma in response to Epo and the EpoR/gammaR1. EpoR/gammaR2 heterodimer is a fully functional receptor complex. The results demonstrate that the configuration of the extracellular domains influences the architecture of the intracellular domains.

Humans↗
PubMed1997

The interferon gamma (IFN-gamma) receptor: a paradigm for the multichain cytokine receptor.

With the purification and cloning of the interferon gamma (IFN-gamma) receptor chains the mechanism of IFN-gamma action and the resultant signal transduction events were delineated in remarkable detail. The interferon gamma (IFN-gamma) receptor complex consists of two chains: IFN-gammaR1, the ligand-binding chain, and IFN-gammaR2, the accessory chain. Binding of IFN-gamma causes oligomerization of the two IFN-gamma receptor subunits, IFN-gammaR1 and IFN-gammaR2, which initiates the signal transduction events: activation of Jak1 and Jak2 receptor associated protein tyrosine kinases, phosphorylation of the IFN-gammaR1 intracellular domain on Tyr440 followed by phosphorylation and activation of Stat1alpha, the latent transcriptional factor. With all these steps established, the IFN-gamma receptor complex has provided the basic model for understanding the receptors for other members of the family of class II cytokine receptors.

Animals↗
PubMed1997

A JAK1/JAK2 chimera can sustain alpha and gamma interferon responses.

Cell lines that are mutated in interferon (IFN) responses have been critical in establishing an essential role for the JAK family of nonreceptor tyrosine kinases in interferon signalling. Mutant gamma1A cells have previously been shown to be complemented by overexpression of JAK2. Here, it is shown that these cells carry a defect in, and can also be complemented by, the beta-subunit of the IFN-gamma receptor, consistent with the hypothesis that the mutation in these cells affects JAK2-receptor association. In contrast, mutant gamma2A cells lack detectable JAK2 mRNA and protein. By using gamma2A cells, the role of various domains and conserved tyrosine residues of JAK2 in IFN-gamma signalling was examined. Individual mutation of six conserved tyrosine residues, mutation of a potential phosphatase binding site, or mutation of the arginine residue in the proposed SH2-like domain had no apparent effect on signalling in response to IFN-gamma. Results with deletion mutants, however, indicated that association of JAK2 with the IFN-gammaR2 subunit requires the amino-terminal region but not the pseudokinase domain. Consistent with this, in chimeras with JAK1, the JAK2 amino-terminal region was required for receptor association and STAT1 activation. Conversely, a JAK1-JAK2 chimera with the amino-terminal domains of JAK1 linked to the pseudokinase and kinase domains of JAK2 is capable of reconstituting JAK-STAT signalling in response to IFN-alpha and -gamma in mutant U4C cells lacking JAK1. The specificity of the JAKs may therefore lie mainly in their structural interaction with different receptor and signalling proteins rather than in the substrate specificity of their kinase domains.

Amino Acid Sequence↗