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T Jayaraman

Publications and source records attributed to T Jayaraman.

18 recordsLinked to original sources
PubMed2000

PKA phosphorylation dissociates FKBP12.6 from the calcium release channel (ryanodine receptor): defective regulation in failing hearts.

The ryanodine receptor (RyR)/calcium release channel on the sarcoplasmic reticulum (SR) is the major source of calcium (Ca2+) required for cardiac muscle excitation-contraction (EC) coupling. The channel is a tetramer comprised of four type 2 RyR polypeptides (RyR2) and four FK506 binding proteins (FKBP12.6). We show that protein kinase A (PKA) phosphorylation of RyR2 dissociates FKBP12.6 and regulates the channel open probability (Po). Using cosedimentation and coimmunoprecipitation we have defined a macromolecular complex comprised of RyR2, FKBP12.6, PKA, the protein phosphatases PP1 and PP2A, and an anchoring protein, mAKAP. In failing human hearts, RyR2 is PKA hyperphosphorylated, resulting in defective channel function due to increased sensitivity to Ca2+-induced activation.

Animals↗
PubMed2000

Calcineurin is downstream of the inositol 1,4,5-trisphosphate receptor in the apoptotic and cell growth pathways.

The inositol 1,4,5-trisphosphate receptor (IP(3)R) is a calcium (Ca(2+)) release channel found on the endoplasmic reticulum of virtually all types of cells. Human T lymphocytes (Jurkat) that are made deficient in IP(3)R do not generate Ca(2+) signals in response to T cell receptor stimulation, fail to translocate the nuclear factor for activated T cells to the nucleus, and are remarkably resistant to induction of apoptosis with CD95 (Fas), dexamethasone, gamma irradiation, and T cell receptor stimulation using anti-CD3 antibody. Expression of constitutively active calcineurin A in IP(3)R-deficient T cells restored nuclear factor for activated T cells translocation to the nucleus and dephosphorylation of Bad and rendered the cells sensitive to apoptotic inducers. Induction of apoptosis required both active calcineurin A (DeltaCnA) and activation-dependent colocalization of CnA with its substrate. Thus, the Ca(2+)-dependent phosphatase calcineurin (CnA) is downstream of the IP(3)R in both the cell growth and apoptotic signaling pathways.

Apoptosis↗
PubMed1999

Inhibition of intimal thickening after balloon angioplasty in porcine coronary arteries by targeting regulators of the cell cycle.

BACKGROUND: Although percutaneous transluminal coronary angioplasty (PTCA) is a highly effective procedure to reduce the severity of stenotic coronary atherosclerotic disease, its long-term success is significantly limited by the high rate of restenosis. Several cellular and molecular mechanisms have been implicated in the development of restenosis post-PTCA, including vascular smooth muscle cell (VSMC) activation, migration, and proliferation. Recently, our group demonstrated that rapamycin, an immunosuppressant agent with antiproliferative properties, inhibits both rat and human VSMC proliferation and migration in vitro. In the present study, we investigated (1) whether rapamycin administration could reduce neointimal thickening in a porcine model of restenosis post-PTCA and (2) the mechanism by which rapamycin inhibits VSMCs in vivo. METHODS AND RESULTS: PTCA was performed on a porcine model at a balloon/vessel ratio of 1.7+/-0.2. Coronary arteries were analyzed for neointimal formation 4 weeks after PTCA. Intramuscular administration of rapamycin started 3 days before PTCA at a dose of 0.5 mg/kg and continued for 14 days at a dose of 0.25 mg/kg. Cyclin-dependent kinase inhibitor (CDKI) p27(kip1) protein levels and pRb phosphorylation within the vessel wall were determined by immunoblot analysis. PTCA in the control group was associated with the development of significant luminal stenosis 4 weeks after the coronary intervention. Luminal narrowing was a consequence of significant neointimal formation in the injured areas. Rapamycin administration was associated with a significant inhibition in coronary stenosis (63+/-3.4% versus 36+/-4.5%; P<0.001), resulting in a concomitant increase in luminal area (1.74+/-0.1 mm2 versus 3. 3+/-0.4 mm2; P<0.001) after PTCA. Inhibition of proliferation was associated with markedly increased concentrations of the p27(kip1) levels and inhibition of pRb phosphorylation within the vessel wall. CONCLUSIONS: Rapamycin administration significantly reduced the arterial proliferative response after PTCA in the pig by increasing the level of the CDKI p27(kip1) and inhibition of the pRb phosphorylation within the vessel wall. Therefore, pharmacological interventions that elevate CDKI in the vessel wall and target cyclin-dependent kinase activity may have a therapeutic role in the treatment of restenosis after angioplasty in humans.

Angioplasty, Balloon, Coronary↗
PubMed1997

T cells deficient in inositol 1,4,5-trisphosphate receptor are resistant to apoptosis.

The type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) calcium release channel is present on the endoplasmic reticulum of most cell types. T lymphocytes which have been made deficient in IP3R1 lack detectable IP3-induced intracellular calcium release and exhibit defective signaling via the T-cell receptor (TCR) (T. Jayaraman, E. Ondriasova, K. Ondrias, D. Harnick, and A. R. Marks, Proc. Natl. Acad. Sci. USA 92:6007-6011, 1995). We now show that IP3R1-deficient T cells are resistant to apoptosis induced by dexamethasone, TCR stimulation, ionizing radiation, and Fas. Resistance to TCR-mediated apoptosis in IP3R1-deficient cells is reversed by pharmacologically raising cytoplasmic calcium levels. TCR-mediated apoptosis can be induced in calcium-free media, indicating that extracellular calcium influx is not required. These findings suggest that intracellular calcium release via the IP3R1 is a critical mediator of apoptosis.

Apoptosis↗
PubMed1996

Regulation of the inositol 1,4,5-trisphosphate receptor by tyrosine phosphorylation.

Tyrosine kinases indirectly raise intracellular calcium concentration ([Ca2+]i) by activating phospholipases that generate inositol 1,4,5-trisphosphate (IP3). IP3 activates the IP3 receptor (IP3R), an intracellular calcium release channel on the endoplasmic reticulum. T cell receptor stimulation triggered a physical association between the nonreceptor protein tyrosine kinase Fyn and the IP3R, which induced tyrosine phosphorylation of the IP3R. Fyn activated an IP3-gated calcium channel in vitro, and tyrosine phosphorylation of the IP3R during T cell activation was reduced in thymocytes from fyn-/- mice. Thus, activation of the IP3R by tyrosine phosphorylation may play a role in regulating [Ca2+]i.

Animals↗
PubMed1995

The inositol 1,4,5-trisphosphate receptor is essential for T-cell receptor signaling.

Antigen-specific activation of T lymphocytes, via stimulation of the T-cell antigen receptor (TCR) complex, is marked by a rapid and sustained increase in the concentration of cytoplasmic free Ca2+ ([Ca2+]i). It has been suggested that the second messenger inositol 1,4,5-trisphosphate (IP3) produced after TCR stimulation binds to the IP3 receptor (IP3R), an intracellular Ca(2+)-release channel, and triggers the increase in [Ca2+]i that activates transcription of the gene for T-cell growth factor interleukin 2 (IL-2). However, the role of the IP3R in T-cell signaling and possibly in plasma membrane Ca2+ influx in T cells remains unproven. Stable transfection of T cells (Jurkat) with antisense type 1 IP3R cDNA prevented type 1 IP3R expression, providing a tool for dissecting the role of IP3 signaling during T-cell activation. T cells lacking type 1 IP3R failed to increase [Ca2+]i or produce IL-2 after TCR stimulation. Moreover, depletion of intracellular Ca2+ stores without TCR activation stimulated Ca2+ influx in cells lacking the type 1 IP3R. These results establish that the type 1 IP3R is required for intracellular Ca2+ release that triggers antigen-specific T-cell proliferation but not for plasma membrane Ca2+ influx.

Antigens, CD↗
PubMed1995

The human type 1 inositol 1,4,5-trisphosphate receptor from T lymphocytes. Structure, localization, and tyrosine phosphorylation.

Inositol 1,4,5-trisphosphate receptors (IP3R) are intracellular calcium release channels involved in diverse signaling pathways. An IP3R is thought to play a role in mobilizing calcium required for activation of T lymphocytes. The IP3R is a tetrameric structure comprised of four approximately 300-kDa subunits encoded by a approximately 10-kilobase mRNA. In the present study we determined the structure of the human type 1 IP3R expressed in T lymphocytes (Jurkats). The IP3R in human T cells had a predicted molecular mass of 308 kDa and was most similar to the non-neuronal form of the rodent type 1 IP3R. Two putative tyrosine phosphorylation sites were identified, one near the amino terminus and one near the putative channel pore at the carboxyl terminus. During T cell activation the IP3R was tyrosine phosphorylated. A site-specific anti-IP3R antibody was used to localize the carboxyl terminus of the IP3R to the cytoplasm in T cells.

Amino Acid Sequence↗
PubMed1995

Inositol 1,4,5-trisphosphate receptor in skeletal muscle: differential expression in myofibres.

The role of inositol 1,4,5-trisphosphate as a second messenger in signal transduction has been well established in many cell types. However, conflicting reports have led to a controversy regarding the role, if any, of inositol 1,4,5-trisphosphate signalling in skeletal muscle. Indeed, expression of the inositol 1,4,5-trisphosphate receptor has not previously been demonstrated in skeletal muscle. In the present study we used in situ hybridization, immunohistochemistry, and [3H]-inositol 1,4,5-trisphosphate binding to demonstrate that rat skeletal muscle fibres contain inositol 1,4,5-trisphosphate receptors. RNAse protection and partial sequencing suggested that the inositol 1,4,5-trisphosphate receptors expressed in skeletal muscle was most similar to the non-neuronal form of the type 1 inositol 1,4,5-trisphosphate receptor. While in situ hybridization showed inositol 1,4,5-trisphosphate receptor mRNA in all types of skeletal myofibres, immunodetectable inositol 1,4,5-trisphosphate receptor protein and specific [3H]-inositol 1,4,5-trisphosphate binding sites were preferentially expressed in slow oxidative (type I) and fast oxidative-glycolytic (type IIA) fibres, but not in fast glycolytic (type IIB) fibres. These findings indicate that an inositol 1,4,5-trisphosphate receptor is preferentially expressed in oxidative fibres of skeletal muscle.

Animals↗
PubMed1995

Rapamycin-FKBP inhibits cell cycle regulators of proliferation in vascular smooth muscle cells.

Multiple growth factors can stimulate quiescent vascular smooth muscle cells to exit from G0 and reenter the cell cycle. The macrolide antibiotic rapamycin, bound to its cytosolic receptor FKBP, is an immunosuppressant and a potent inhibitor of cellular proliferation. In the present study, the antiproliferative effects of rapamycin on human and rat vascular smooth muscle cells were examined and compared with the effects of a related immunosuppressant, FK520. In vascular smooth muscle cells, rapamycin, at concentrations as low as 1 ng/mL, inhibited DNA synthesis and cell growth. FK520, an analogue of the immunosuppressant FK506, is structurally related to rapamycin and binds to FKBP but did not inhibit vascular smooth muscle cell growth. Molar excesses of FK520 blocked the antiproliferative effects of rapamycin, indicating that the effects of rapamycin required binding to FKBP. Rapamycin-FKBP inhibited retinoblastoma protein phosphorylation at the G1/S transition. This inhibition of retinoblastoma protein phosphorylation was associated with a decrease in p33cdk2 kinase activity. These observations suggest that rapamycin, but not FK520, inhibits vascular smooth muscle cell proliferation by reducing cell-cycle kinase activity.

Animals↗
PubMed1994

Localization of the high and low affinity [3H]ryanodine binding sites on the skeletal muscle Ca2+ release channel.

The Ca2+ release channel of skeletal muscle sarcoplasmic reticulum is modulated in a biphasic manner by the plant alkaloid ryanodine and there are two distinct binding sites on this channel for ryanodine. The Ca2+ release channel is a homotetramer with a subunit of 5037 amino acids. The ability of sarcoplasmic reticulum membranes to bind [3H]ryanodine to the high affinity site is lost upon proteolysis with trypsin. [3H]Ryanodine, however, bound before proteolysis remains bound after trypsin digestion. If the high affinity site is first occupied with [3H]ryanodine and then 100 microM ryanodine is added to occupy the low affinity sites, almost all of [3H]ryanodine bound to the high affinity site remains bound after proteolysis. Proteolysis causes the solubilized Ca2+ release channel containing bound [3H]ryanodine to undergo four discrete shifts in sedimentation (30 S-->28 S-->26 S-->19 S-->14 S). Polypeptides having apparent molecular masses of 76, 66, 56, 45, 37, and 27 kDa can be identified in the 14 S complex. The 76-, 56-, 45-, and 27-kDa polypeptides have been partially sequenced from the NH2 terminus. In addition, the 76-, 66-, and 27-kDa fragments are recognized by an antibody to the last 9 amino acids at the carboxyl terminus of the skeletal muscle ryanodine receptor and the 76-, 66-, and 37-kDa fragments are recognized by an antibody to a peptide matching the sequence 4670-4685. The 56-kDa and the 45-kDa fragments are not Ca2+ release channel fragments. Both high and low affinity ryanodine binding sites are found in the 14 S complex and are, therefore, most likely located between Arg-4475 and the carboxyl terminus.

Amino Acid Sequence↗
PubMed1994

Stabilization of calcium release channel (ryanodine receptor) function by FK506-binding protein.

FK506-binding protein (FKBP12) was originally identified as the cytosolic receptor for the immunosuppressant drugs FK506 and rapamycin. The cellular function of FKBP12, a ubiquitously expressed 12,000-dalton proline isomerase, has been unknown. FKBP12 copurifies with the 565,000-dalton ryanodine receptor (RyR), four of which form intracellular Ca2+ release channels of the sarcoplasmic and endoplasmic reticula. By coexpressing the RyR and FKBP12 in insect cells, we have demonstrated that FKBP12 modulates channel gating by increasing channels with full conductance levels (by > 400%), decreasing open probability after caffeine activation (from 0.63 +/- 0.09 to 0.04 +/- 0.02), and increasing mean open time (from 4.4 +/- 0.6 ms to 75 +/- 41 ms). FK506 or rapamycin, inhibitors of FKBP12 isomerase activity, reverse these stabilizing effects. These results provide the first natural cellular function for FKBP12, and establish that the functional Ca2+ release channel complex includes FKBP12.

Animals↗
PubMed1994

The ryanodine receptor from canine heart sarcoplasmic reticulum is associated with a novel FK-506 binding protein.

We find that the purified ryanodine receptor/calcium release channel (CRC) isolated from canine heart sarcoplasmic reticulum (SR) is tightly associated with an FK-506 binding protein which is distinct from human recombinant FKBP-12. Fractions from both skeletal and heart muscle were probed by Western Blot analysis using sequence specific antibody raised against the N-terminus of human recombinant FKBP-12. Only FKBP-12 was detected in rabbit and dog skeletal muscle SR as well as in dog heart cytosol. Canine heart SR and the purified canine heart ryanodine receptor contained an immunoreactive band with a somewhat slower relative mobility than that of human recombinant FKBP-12. These observations indicate that the association between the cardiac CRC and this novel FK-506 binding protein is specific.

Animals↗
PubMed1993

Rapamycin-FKBP12 blocks proliferation, induces differentiation, and inhibits cdc2 kinase activity in a myogenic cell line.

Rapamycin is a potent immunosuppressant that binds to the cytosolic protein, FKBP12, and blocks T cell activation. Here we report that rapamycin also blocks myogenic proliferation and induces differentiation, associated with a decrease in p34cdc2 activity and cyclin A levels. In yeast and mammals, rapamycin blocks cell cycle progression by causing G1 arrest, arguing for a conserved signaling pathway governing the G1 to S transition. p34cdc2 has been shown to play a role in both the transition from G1 to S and from G2 to M in yeast. In higher eukaryotes the role of p34cdc2 in G1 to S transition is less clear. Rapamycin and the structurally related macrolide antibiotic FK506 both bind to a cytosolic protein, the FK506-binding protein (FKBP12). We show that inhibition of myogenic proliferation is achieved at low doses of rapamycin (< 1 ng/ml) and is competed by a molar excess of FK506, indicating specificity for FKBP12. The distinct FK506-calcineurin pathway did not affect myogenic proliferation, differentiation, or p34cdc2 kinase activity. Thus, the rapamycin-FKBP12 signaling pathway involves a specific and direct effect on p34cdc2 kinase activity at the G1 to S transition and identifies a regulatory step during myogenic differentiation.

Animals↗
PubMed1992

FK506 binding protein associated with the calcium release channel (ryanodine receptor).

The calcium release channel (CRC)/ryanodine receptor (RyRec) has been identified as the foot structure of the sarcoplasmic reticulum (SR) and provides the pathway for calcium efflux required for excitation-contraction coupling in skeletal muscle. The CRC has previously been reported to consist of four identical 565-kDa protomers. We now report the identification of a 12-kDa protein which is tightly associated with highly purified RyRec from rabbit skeletal muscle SR. N-terminal amino acid sequencing and cDNA cloning demonstrates that the 12-kDa protein from fast twitch skeletal muscle is the binding protein for the immunosuppressant drug FK506. In humans, FK506 binds to the 12-kDa FK506-binding protein (FKBP12) and blocks calcium-dependent T cell activation. We find that FKBP12 and the RyRec are tightly associated in skeletal muscle SR on the basis of: 1) co-purification through sequential heparin-agarose, hydroxylapatite, and size exclusion chromatography columns; 2) coimmunoprecipitation of the RyRec and FKBP12 with anti-FKBP12 antibodies; and 3) subcellular localization of both proteins to the terminal cisternae of the SR, and not in the longitudinal tubules of SR, in fast twitch skeletal muscle. The molar ratio of FKBP12 to RyRec in highly purified RyRec preparations is approximately 1:4, indicating that one FKBP12 molecule is associated with each calcium release channel/foot structure.

Amino Acid Sequence↗
PubMed1984

Evidence for the presence of M. leprae reactive T lymphocytes in patients with lepromatous leprosy.

Evidence for the presence of Mycobacterium leprae reactive T cells in many lepromatous leprosy (LL) patients was obtained using in vitro antigen-induced lymphoproliferative responses. (1) Co-cultures of T enriched cells from LL patients when combined with 2 h adherent cells (AC) from HLA-D compatible tuberculoid leprosy individuals showed significant levels of 3H-thymidine incorporation in the presence of soluble and integral M. leprae antigens. (2) More interestingly, autologous T cell + AC co-cultures also showed significant improvement in antigen-induced lymphoproliferation in nine of 16 lepromatous patients. Insignificant improvement was observed in similar co-cultures of tuberculoid leprosy patients. (3) Addition of exogenous, purified human interleukin-2 (IL-2) to antigen stimulated PBMC from some lepromatous patients showed the best improvement in terms of overall 3H-thymidine incorporation, indicating that lepromatous patients possess T cells which can differentiate to an IL-2 responsive state. Significantly, the level of proliferation varied within the group. A proportion of clinically similar lepromatous patients failed to show improvement by any of the above methods.

Antigens, Bacterial↗