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Biomedical subjects

I Chackalaparampil

Publications and source records attributed to I Chackalaparampil.

15 recordsLinked to original sources
PubMed2001

Adenovirus DNA binding protein interacts with the SNF2-related CBP activator protein (SrCap) and inhibits SrCap-mediated transcription.

The SNF2-related CBP activator protein, SrCap (pronounced "sir cap"), shares homology with the SNF2/SWI2 protein family. SrCap was cloned through its ability to bind CBP. SrCap can function as a CBP coactivator and can activate transcription in a reporter assay when expressed as a Gal-SrCap fusion protein. A monoclonal antibody raised against the carboxyl terminus of SrCap coimmunoprecipitates CBP/p300, supporting the model that SrCap is a CBP binding protein and that these proteins can be found together in a cellular protein complex. In addition, several cellular proteins are coimmunoprecipitated by the SrCap-specific antibody. Since adenovirus E1A proteins interact with CBP/p300 proteins, we examined what proteins could be copurified in a SrCap-specific coimmunoprecipitation assay from lysates of adenovirus-infected cells. While E1A proteins were not detected in this complex, to our surprise, we observed the presence of an infected-cell-specific band of 72 kDa, which we suspected might be the adenovirus DNA binding protein, DBP. The adenovirus DBP is a multifunctional protein involved in several aspects of the adenovirus life cycle, including an ability to modulate transcription. The identity of DBP was confirmed by DBP-specific Western blot analysis and by reimmunoprecipitating DBP from denatured SrCap-specific protein complexes. Using in vitro-translated DBP and SrCap proteins, we demonstrated that these proteins interact. To determine whether this interaction could affect SrCap-mediated transcription, we tested whether increasing amounts of DBP could modulate the Gal-SrCap transcription activity. We observed that DBP inhibited Gal-SrCap transcription activity in a dose-dependent manner. These data suggest a novel mechanism of adenovirus host cell control by which DBP binds to and inactivates SrCap, a member of the SNF2 chromatin-remodeling protein family.

Adenosine Triphosphatases↗
PubMed1999

Identification of a novel SNF2/SWI2 protein family member, SRCAP, which interacts with CREB-binding protein.

The ability of cAMP response-element binding protein (CREB)-binding protein (CBP) to function as a co-activator for a number of transcription factors appears to be mediated by its ability to act as a histone acetyltransferase and through its interaction with a number of other proteins (general transcription factors, histone acetyltransferases, and other co-activators). Here we report that CBP also interacts with a novel ATPase termed Snf2-Related CBP Activator Protein (SRCAP). Consistent with this activity, SRCAP contains the conserved ATPase domain found within members of the Snf2 family. Transfection experiments demonstrate that SRCAP is able to activate transcription when expressed as a Gal-SRCAP chimera and that SRCAP also enhances the ability of CBP to activate transcription. The adenoviral protein E1A was found to disrupt interaction between SRCAP and CBP possibly representing a mechanism for E1A-mediated transcriptional repression.

Adenosine Triphosphatases↗
PubMed1997

Potential roles of osteopontin and alphaVbeta3 integrin in the development of coronary artery restenosis after angioplasty.

Angioplasty procedures are increasingly used to reestablish blood flow in blocked atherosclerotic coronary arteries. A serious complication of these procedures is reocclusion (restenosis), which occurs in 30-50% of patients. Migration of coronary artery smooth muscle cells (CASMCs) to the site of injury caused by angioplasty and subsequent proliferation are suggested mechanisms of reocclusion. Using both cultured human CASMCs and coronary atherectomy tissues, we studied the roles of osteopontin (OPN) and one of its receptors, alphavbeta3 integrin, in the pathogenesis of coronary restenosis. We also measured the plasma levels of OPN before and after angioplasty and determined the effect of exogenous OPN on CASMC migration, extracellular matrix invasion, and proliferation. We found that cultured CASMCs during log phase of growth and smooth muscle cell layer of the coronary atherosclerotic tissues of patients express both OPN mRNA and protein at a significantly elevated level compared with controls. Interestingly, whereas the baseline plasma OPN levels in control samples were virtually undetectable, those in patient plasma were remarkably high. We also found that interaction of OPN with alphavbeta3 integrin, expressed on CASMCs, causes migration, extracellular matrix invasion, and proliferation. These effects were abolished when OPN or alphavbeta3 integrin gene expression in CASMCs was inhibited by specific antisense S-oligonucleotide treatment or OPN-alphavbeta3 interaction was blocked by treatment of CASMCs with antibodies against OPN or alphavbeta3 integrin. Our results demonstrate that OPN and alphavbeta3 integrin play critical roles in regulating cellular functions deemed essential for restenosis. In addition, these results raise the possibility that transient inhibition of OPN gene expression or blocking of OPN-alphavbeta3 interaction may provide a therapeutic approach to preventing restenosis.

Adolescent↗
PubMed1997

Altered sialylation of osteopontin prevents its receptor-mediated binding on the surface of oncogenically transformed tsB77 cells.

It has been reported previously that oncogenically transformed cells secrete different molecular forms of osteopontin (OPN), a sialic acid-rich, adhesive, phosphoglycoprotein, than OPNs secreted by their nontransformed counterparts. However, the origin of the OPN isoform secreted by the transformed cells and whether it has different physiological properties which may serve transformation-specific functions remain poorly understood. Here, we report that Rat-1 cells transformed by a temperature-sensitive mutant of Rous sarcoma virus (tsB77) secrete two discrete molecular forms of OPN, a 69-kDa OPN at the nonpermissive temperature (41 degrees C) and a 62-kDa form at the permissive temperature (34 degrees C). However, tsB77 cells at both temperatures transcribe a single 1.6 kb OPN mRNA and contain only the 69-kDa form of OPN intracellularly, suggesting that the 69-kDa OPN is modified to the 62-kDa form prior to or immediately after secretion by cells at 34 degrees C. We ruled out proteolytic cleavage, differential phosphorylation, or lack of N- or O-linked carbohydrates as the possible mechanism, but found that the 62-kDa OPN contains significantly reduced levels of sialic acid, as compared to its 69-kDa form. The binding assays using 32P-labeled OPN revealed that only the 69-kDa OPN, not its 62-kDa form, undergoes receptor-mediated localization on the cell surface, although tsB77 cells synthesize OPN receptors (alpha(v)beta3 integrins) at both permissive and nonpermissive temperatures. Furthermore, 125I-labeled purified milk OPN, which is highly sialylated and shows cell surface binding, upon digestion with neuraminidase failed to interact with the cell surface. Taken together, these results suggest that the difference between the 69-kDa and 62-kDa isoforms of OPN resides in their sialic acid content, and sialylation of OPN is crucial for its receptor-mediated binding on tsB77 cells. The data presented here demonstrate for the first time a physiological role of sialic acids in this protein, and raise the possibility that oncogenically transformed tsB77 cells may exploit the lack of OPN-receptor interactions for their invasive behavior.

Animals↗
PubMed1996

Cells in vivo and in vitro from osteopetrotic mice homozygous for c-src disruption show suppression of synthesis of osteopontin, a multifunctional extracellular matrix protein.

Mice carrying homozygous disruption of the c-src proto-oncogene (Src-/-) develop osteopetrosis due to an impaired ability of osteoclasts to adhere to the bone surface and/or to form bone-resorbing ruffled border. It has also been reported that osteopontin (OPN), a secreted phosphoprotein, mediates osteoclast adherence to the bone matrix. We report here that cells from Src-/- mice, both in vitro and in vivo, express OPN mRNA and protein at a significantly reduced level as compared to cells from Src+/- and +/+ animals, suggesting a potential role for the proto-oncogene c-src in the regulation of OPN gene expression. Our data also show that OPN gene expression can be induced by treatment of SR-/- cells with epidermal growth factor (EGF) and 12-O-tetradecanoyl phorbol-13-acetate (TPA). Results obtained from studies using inhibitors of receptor tyrosine kinases (RTKs) and protein kinase C (PKC) suggest that PKC and RTK are positioned in a pathway with PKC as the downstream effector for the EGF-induced OPN gene expression in SRC-/- cells, and that pp60c-src and EGF may regulate OPN gene expression through a common signalling pathway. Furthermore, contrary to published reports, our study shows that EGF-mediated cell signalling does not require functional interaction between the EGF-receptor and pp60c-src.

3T3 Cells↗
PubMed1994

Osteopontin: its transglutaminase-catalyzed posttranslational modifications and cross-linking to fibronectin.

Osteopontin (OP) is a component of extracellular, bone, and urinary stone matrices, but the mechanism by which it is stably incorporated into such matrices remains unknown. By SDS-PAGE analysis of [125I]OP, treated with a catalytic amount of TG, we first demonstrate both intra- and intermolecular covalent cross-linking of OP. Most importantly, the analysis of the products generated from reactions containing OP, Fn, and TG by SDS-PAGE, autoradiography, and Western blotting using either OP or Fn antibody, and quantitation of TG-catalyzed epsilon-(gamma-glutamyl)lysine isopeptide formation between OP and Fn demonstrate, for the first time, covalent cross-linking between these two proteins. Similar reactions in the presence of polyamine substrates of TG show OP-Fn intermolecular cross-linking via N,N-bis-(gamma-glutamyl)polyamine formation. Finally, immunoprecipitation of 125I-labeled NRK cell surface proteins with anti-OP and anti-Fn antibodies, SDS-PAGE analysis, and autoradiography provides critical evidence for nonreducible OP-Fn cross-linking in vivo. These results clearly suggest that TG-mediated cross-linking between OP and Fn represents one of the most likely mechanisms by which OP becomes covalently linked to bone matrix, urinary stone matrix, and to ECM.

Amino Acid Sequence↗
PubMed1994

Tyrosine dephosphorylation of pp60c-src is stimulated by a serine/threonine phosphatase inhibitor.

Incubation of NIH3T3-derived c-src overexpressor cells with okadaic acid, a specific serine/threonine phosphatase inhibitor, stimulates pp60c-src kinase activity about 2-3-fold. Activation is blocked if cells are simultaneously treated with orthovanadate, a tyrosine phosphatase inhibitor. Furthermore, okadaic acid treatment induces a small decrease in Tyr 527 phosphorylation of wild-type pp60c-src and a large decrease in Tyr 527 phosphorylation of kinase-defective pp60c-src(Lys 295-->Arg). These results suggest that the activation is mediated by okadaic acid-induced changes in tyrosine phosphorylation of pp60c-src involving 'cross-over' from serine/threonine to tyrosine signal transduction pathways. Stimulation of pp60c-src activity and Tyr 527 dephosphorylation do not require changes in serine/threonine phosphorylation of pp60c-src, suggesting that these changes result from modulation of an upstream Tyr 527 phosphatase or kinase which is itself regulated by altered serine/threonine phosphorylation. Since okadaic acid induces a pseudo-mitotic phenotype in rodent cells (K. Yamashita, H. Yasuda, J. Pines, K. Yasumoto, H. Nishitani, M. Ohtsubo, T. Hunter, T. Sugimura and T. Nishimoto, EMBO J., 9: 4331-4338, 1990), it is possible that these phenomena are induced by a biochemical mechanism similar to that which causes transient tyrosine dephosphorylation of pp60c-src during mitosis.

3T3 Cells↗
PubMed1992

Role of p34cdc2-mediated phosphorylations in two-step activation of pp60c-src during mitosis.

Phosphorylation of pp60c-src by p34cdc2 at three amino-proximal serine/threonine residues is temporally correlated with, but insufficient for, mitotic activation of c-Src kinase. The direct cause of activation during mitosis appears to be temporally correlated partial dephosphorylation of Tyr-527, a residue whose phosphorylation strongly suppresses pp60c-src activity. Site-directed mutagenesis of the serine/threonine phosphorylation sites blocks half the mitosis-specific decrease in Tyr-527 phosphorylation and half the increase in pp60c-src kinase activity. We conclude that p34cdc2 partially activates pp60c-src by a two-step process in which its serine/threonine phosphorylations either sensitize pp60c-src to a Tyr-527 phosphatase or desensitize it to a Tyr-527 kinase. Furthermore, additional events, independent of these p34cdc2-mediated phosphorylations, participate in mitotic activation of pp60c-src.

3T3 Cells↗
PubMed1992

c-Src and mitosis.

The transforming potential and by inference the physiological function of the proto-oncoprotein pp60c-src closely correlate with the level of its protein tyrosine kinase activity. We have investigated the cell cycle-dependent regulation of this activity using mouse fibroblasts overexpressing chicken or mouse pp60c-src as a model system. During mitosis pp60c-src becomes phosphorylated at specific serine and threonine residues by p34cdc2. At the same time its tyrosine kinase activity, assayed in vitro, is increased approximately twofold and accessibility of its SH2 domain for binding relevant phosphotyrosine-containing ligands increases by about 15-fold. A kinase-defective mutant of pp60c-src exhibits a substantial (50-70%) decrease in phosphorylation at Tyr527 during mitosis. Phosphorylation of this residue negatively regulates kinase activity. Indirect evidence indicates a lesser decrease in wild-type pp60c-src Tyr527 phosphorylation during mitosis. Coordinate mutation of the mitosis-specific phosphorylation (MSP) sites in kinase-defective pp60c-src greatly reduces, though does not abolish, its mitosis-specific tyrosine dephosphorylation. Similarly, coordinate mutation of the three MSP sites in chicken pp60c-src or the corresponding two sites in mouse pp60c-src does not completely block mitotic stimulation of kinase activity. Thus, additional events beyond p34cdc2-mediated phosphorylation are involved in cell-cycle dependent regulation of pp60c-src activity. This is also suggested by the stimulation of pp60c-src kinase activity and decrease in phosphorylation of Tyr527 observed following treatment of fibroblasts with okadaic acid, a potent inhibitor of types 1 and 2A serine/threonine phosphatases. The potential role of cell cycle-dependent regulation of phosphatases and kinases acting on the regulatory tyrosine residue of pp60c-src is discussed.

Animals↗
PubMed1991

Altered tyrosine 527 phosphorylation and mitotic activation of p60c-src.

The tyrosine kinasee activity of p60c-src, the protein product of the c-src gene, increases during mitosis; this may be important in initiating at least some of the cellular changes that occur during this phase of the cell cycle. Although there is evidence that p60c-src is phosphorylated at several sites during mitosis, phosphorylation in vitro does not increase its kinase activity. We now report that the kinase activity of a p60c-src mutant with residue tyrosine 527 changed to phenylanine does not change during the cell cycle, suggesting that changes in the phosphorylation state of this residue may be responsible for the activation of p60c-src at mitosis. Although changes in phosphorylation at Tyr 527 cannot be detected with the wild-type protein we find that phosphorylation at Tyr 527 of a mutant with reduced kinase activity decreases threefold during mitosis. On the basis of these results we suggest that activation of p60c-src at mitosis results from decreased phosphorylation on Tyr 527, and that p60c-src may be or may activate the kinase that phosphorylates Tyr 527.

Animals↗
PubMed1988

Altered phosphorylation and activation of pp60c-src during fibroblast mitosis.

At least half the pp60c-src in NIH 3T3-derived c-src overexpresser cells in modified by novel threonine and, possibly, serine phosphorylation within its amino 16 kd region during mitosis. At the same time, the tryptic phosphopeptide containing Ser 17, the site of cyclic AMP-dependent phosphorylation, is either modified or dephosphorylated. While the amount of pp60c-src is not significantly altered, the in vitro-specific kinase activity of modified pp60c-src is enhanced 4- to 7-fold. Modified pp60c-src has the same tyrosine-containing tryptic phosphopeptides as pp60c-src from unsynchronized cells, indicating that activation is independent of Tyr 416/Tyr 527 phosphorylation. Electrophoretic mobility retardations indicated that endogenous pp60c-src and pp60v-src are similarly modified during mitosis. The modifications and enhanced activity disappear near the time of cell division. These results suggest that pp60c-src is regulated by and, in turn, may regulate mitosis-specific events in fibroblasts.

Animals↗
PubMed1987

Epidermal and transforming growth factors modulate secretion of a 69 kDa phosphoprotein in normal rat kidney fibroblasts.

Our study shows that the secretion of a major non-glycosylated, phosphoprotein of 69 kDa (pp69) is a specific marker for non-transformed NRK-49F cells. Treatment of NRK-49F cells with EGF alone or with different combinations of EGF plus TGF-beta modulates the secretion of pp69, suggesting its relationship with cellular proliferation. Antibody raised against pp69 recognizes, in addition to pp69, another major phospho-protein of 62 kDa (pp62) secreted by RR1022 and spontaneously transformed NRK-49F cells. Immunoprecipitation of total cell lysates from both NRK-49F and RR1022 cells with anti-pp69 antibody detected only pp69. These observations suggest a precursor-product relationship between pp69 secreted by non-transformed NRK-49F cells and pp62 secreted by transformed cells.

Animals↗
PubMed1986

Ability of oncogenically transformed cells to grow without anchorage correlates with phosphorylation of a group of cell surface membrane proteins.

Anchorage-independent growth in vitro is strongly correlated with cellular malignancy in vivo and it has been shown that retinoic acid (RA; a vitamin A analog) inhibits anchorage-independent growth of a wide variety of oncogenically transformed cells (RA-sensitive cells). We report here that decreased or lack of phosphorylation of a group of low molecular weight (20-30 kD) cell surface membrane proteins, particularly one of Mr 28 kD, correlates strongly with RA-induced loss of anchorage-independent growth of RA-sensitive cells. Our studies also show that this group of proteins are not phosphorylated in non-transformed cells which do not grow in an anchorage-independent manner. Analysis of [35S]methionine-labeled proteins revealed that these polypeptides are present in both RA-treated and untreated cell surface membranes. This suggests that modulation of phosphorylation rather than lack of synthesis of these proteins is correlated with anchorage regulation of cells. V8 protease mapping of the 28 kD phosphoprotein from transformed cells, irrespective of their origin or of transforming agents, revealed complete fragment homology. Furthermore, the 28 kD phosphoprotein was found to be phosphorylated exclusively at threonine residues. The data obtained from this study suggest that the ability of cells to grow without anchorage is correlated with the phosphorylation of a group of cell surface membrane proteins and RA inhibits anchorage-independent growth by interfering with the phosphorylation rather than synthesis of these proteins.

Animals↗
PubMed1985

Altered processing of a major secreted phosphoprotein correlates with tumorigenicity in Rous sarcoma virus-transformed mammalian cells.

Anchorage-independent growth is highly correlated with neoplastic growth in vivo, and the retinoids (vitamin A and its analogs) inhibit this property in a wide variety of oncogenically transformed cells. We report here that retinoic acid-treated Rous sarcoma virus-transformed rat (RR1022) and vole (SR-1T) cells, which show reversible loss of anchorage-independent growth and assume nontransformed morphology, secrete a major 69-kilodalton phosphoprotein (pp69) instead of the 62-kilodalton phosphoprotein (pp62) secreted by their untreated counterparts. As determined by V8 protease mapping and by two-dimensional electrophoretic analysis, this 69-kilodalton polypeptide was indistinguishable from the pp69 released by nontransformed normal rat kidney cells. Neither retinoic acid-treated RR1022 cells nor normal rat kidney cells secreted pp62, and retinoic acid treatment did not have any significant effect on the synthesis, subcellular localization, or phosphokinase activity of pp60src. Furthermore, treatment with retinoic acid did not alter the synthesis of the transformation-specific 53-kilodalton phosphoprotein (p53) and secretion of the transforming growth factors in RR1022 cells. Our studies showed that there is a clear correlation between the release of pp69 or pp62 and the ability of cells to grow in vitro with or without anchorage. This may provide an important clue for elucidating specific biochemical events involved in anchorage regulation of growth.

Animals↗