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Biomedical subjects

D A Fruman

Publications and source records attributed to D A Fruman.

At least 19 recordsLinked to original sources
PubMed2004

Towards an understanding of isoform specificity in phosphoinositide 3-kinase signalling in lymphocytes.

The PI3K (phosphoinositide 3-kinase) signalling pathway promotes proliferation and transformation in many cell types. This is particularly well illustrated by studies of primary lymphocytes and their leukaemic counterparts. PI3K activation is required for proliferation of T cells and B cells, and certain oncogenes cause leukaemia in part by promoting PI3K signalling. Genetic manipulation of this pathway, together with biochemical studies in primary lymphocytes, has begun to shed light on the molecular mechanisms by which PI3K contributes to the proliferation of normal and transformed lymphocytes. In particular, targeted gene disruption in mice has allowed the identification of specific isoforms of PI3K that are required for distinct cellular responses. Continued investigation of isoform specificity in PI3K signalling, as well as the characterization of critical downstream targets of PI3K signalling, may reveal strategies for the therapeutic control of immune responses and leukaemia.

Animals↗
PubMed2001

Phosphatidylinositol 3-kinase confers resistance to encephalomyocarditis and herpes simplex virus-induced cell death through the activation of distinct downstream effectors.

The Janus kinase/STAT pathway has emerged as the paradigm of IFN-induced protection from viral infections. However, the possible participation of other signaling proteins in this protection is not clearly understood. In this report, we demonstrate that activation of phosphatidylinositol 3-kinase (PI3K) by either serum factors or IFNs blocks cell death induced by encephalomyocarditis virus (EMCV) and HSV. This increased resistance to virus-induced cell death does not involve the activation of the STAT pathway and occurs in the presence of normal viral replication. Interestingly, the cell uses two different PI3K regulated pathways to block EMCV- and HSV-induced cell death. The increased sensitivity of p85alpha(-/-) embryonic fibroblasts to EMCV-induced cell death is specifically corrected by overexpression of an activated allele of Akt/protein kinase B, but not activated mitogen-activated protein kinase extracellular kinase. Conversely, the augmented sensitivity of p85alpha(-/-) cells to HSV-induced cell death was compensated for by expression of an activated form of mitogen-activated protein kinase extracellular kinase, but not by activated Akt/protein kinase B. We conclude from these data that PI3K-activated pathways function in parallel with the Janus kinase/STAT pathway to protect cells from the lethal effects of viruses.

Animals↗
PubMed2000

Impaired kit- but not FcepsilonRI-initiated mast cell activation in the absence of phosphoinositide 3-kinase p85alpha gene products.

The class I(A) phosphoinositide 3-kinases (PI3Ks) consist of a 110-kDa catalytic domain and a regulatory subunit encoded by the p85alpha, p85beta, or p55gamma genes. We have determined the effects of disrupting the p85alpha gene on the responses of mast cells stimulated by the cross-linking of Kit and FcepsilonRI, receptors that reflect innate and adaptive responses, respectively. The absence of p85alpha gene products partially inhibited Kit ligand/stem cell factor-induced secretory granule exocytosis, proliferation, and phosphorylation of the serine/threonine kinase Akt. In contrast, p85alpha gene products were not required for FcepsilonRI-initiated exocytosis and phosphorylation of Akt. LY294002, which inhibits all classes of PI3Ks, strongly suppressed Kit- and FcepsilonRI-induced responses in p85alpha -/- mast cells, revealing the contribution of another PI3K family member(s). In contrast to B lymphocytes, mast cell proliferation was not dependent on Bruton's tyrosine kinase, a downstream effector of PI3K, revealing a distinct pathway of PI3K-dependent proliferation in mast cells. Our findings represent the first example of receptor-specific usage of different PI3K family members in a single cell type. In addition, because Kit- but not FcepsilonRI-initiated signaling is associated with mast cell proliferation, the results provide evidence that distinct biologic functions signaled by these two receptors may reflect differential usage of PI3Ks.

Animals↗
PubMed2000

A peptide library approach identifies a specific inhibitor for the ZAP-70 protein tyrosine kinase.

We utilized a novel peptide library approach to identify specific inhibitors of ZAP-70, a protein Tyr kinase involved in T cell activation. By screening more than 6 billion peptides oriented by a common Tyr residue for their ability to bind to ZAP-70, we determined a consensus optimal peptide. A Phe-for-Tyr substituted version of the peptide inhibited ZAP-70 protein Tyr kinase activity by competing with protein substrates (K(I) of 2 microM). The related protein Tyr kinases, Lck and Syk, were not significantly inhibited by the peptide. When introduced into intact T cells, the peptide blocked signaling downstream of ZAP-70, including ZAP-70-dependent gene induction, without affecting upstream Tyr phosphorylation. Thus, screening Tyr-oriented peptide libraries can identify selective peptide inhibitors of protein Tyr kinases.

Amino Acid Sequence↗
PubMed2000

Hypoglycaemia, liver necrosis and perinatal death in mice lacking all isoforms of phosphoinositide 3-kinase p85 alpha.

Phosphoinositide 3-kinases produce 3'-phosphorylated phosphoinositides that act as second messengers to recruit other signalling proteins to the membrane. Pi3ks are activated by many extracellular stimuli and have been implicated in a variety of cellular responses. The Pi3k gene family is complex and the physiological roles of different classes and isoforms are not clear. The gene Pik3r1 encodes three proteins (p85 alpha, p55 alpha and p50 alpha) that serve as regulatory subunits of class IA Pi3ks (ref. 2). Mice lacking only the p85 alpha isoform are viable but display hypoglycaemia and increased insulin sensitivity correlating with upregulation of the p55 alpha and p50 alpha variants. Here we report that loss of all protein products of Pik3r1 results in perinatal lethality. We observed, among other abnormalities, extensive hepatocyte necrosis and chylous ascites. We also noted enlarged skeletal muscle fibres, brown fat necrosis and calcification of cardiac tissue. In liver and muscle, loss of the major regulatory isoform caused a great decrease in expression and activity of class IA Pi3k catalytic subunits; nevertheless, homozygous mice still displayed hypoglycaemia, lower insulin levels and increased glucose tolerance. Our findings reveal that p55 alpha and/or p50 alpha are required for survival, but not for development of hypoglycaemia, in mice lacking p85 alpha.

Abnormalities, Multiple↗
PubMed1999

SYK is upstream of phosphoinositide 3-kinase in B cell receptor signaling.

We have recently demonstrated that the D3-phosphoinositide phosphatidylinositol 3,4,5-trisphosphate (PtdIns-3,4,5-P(3)) is critical for producing sustained calcium signals through its role in promoting the function of TEC family tyrosine kinases such as Bruton's tyrosine kinase. Although PtdIns-3,4,5-P(3) can potentially be synthesized by any of several types of phosphoinositide 3-kinases (PI3Ks), B cell receptor (BCR)-induced PtdIns-3,4,5-P(3) production is thought to occur primarily through the activation of the class Ia (p85/p110) PI3Ks. This process has been proposed to be mediated by an interaction between the Src family kinase LYN and the p85 subunit of PI3K and/or through p85 membrane recruitment mediated by CBL and/or CD19. However, calcium signaling and other PI3K-dependent signals are relatively preserved in a LYN kinase-deficient B lymphocyte cell line, suggesting that an alternative pathway for PI3K activation exists. As SYK/ZAP70 kinases are upstream from many BCR-initiated signaling events, we directly analyzed SYK-dependent accumulation of both PtdIns-3,4,5-P(3) and PtdIns-3,4-P(2) in B cell receptor signaling using both dominant negative and genetic knockout approaches. Both methods indicate that SYK is upstream of, and necessary for, a significant portion of BCR-induced PtdIns-3,4, 5-P(3) production. Whereas CD19 does not appear to be involved in this SYK-dependent pathway, the SYK substrate CBL is likely involved as the dominant negative SYK markedly attenuates CBL tyrosine phosphorylation and completely blocks the BCR-dependent association of CBL with p85 PI3K.

Animals↗
PubMed1999

Mouse phosphoinositide 3-kinase p110alpha gene: cloning, structural organization, and localization to chromosome 3 band B.

Phosphoinositide 3-Kinases (PI3-Kinases) are a family of dual specificity enzymes with a unique lipid kinase activity toward the D-3 position of the inositol ring of phosphoinositides and a less well characterized serine/threonine protein kinase activity. Class IA PI3-Kinases comprise a 110-120 kDa catalytic subunit (usually termed p110) and an 85 kDa or 50 to 55 kDa regulatory subunit (often called p85). cDNAs for three mammalian Class IA PI3-Kinase catalytic subunits designated p110alpha, p110beta, and p110delta have been cloned from several species. A YAC clone for the human p110alpha gene has also been cloned and mapped to chromosome 3q26.3. However, structural organization for any of the PI3-Kinase p110alpha genes has not been reported. Here, we report the cloning, structural organization, and chromosomal localization of the mouse PI3-Kinase p110alpha gene. The translated portion of the mouse p110alpha gene is encoded by 19 exons that span at least 24 kb. Dual color fluorescence in situ hybridization (FISH) was performed to determine the chromosomal localization of the mouse PI3-Kinase p110alpha gene. FISH results and DAPI banding demonstrated localization of the p110alpha gene to band B on mouse chromosome 3, a region syntenic with human chromosome 3q26.3.

Animals↗
PubMed1999

The SH2 domain-containing inositol 5'-phosphatase (SHIP) recruits the p85 subunit of phosphoinositide 3-kinase during FcgammaRIIb1-mediated inhibition of B cell receptor signaling.

Coligation of FcgammaRIIb1 with the B cell receptor (BCR) or FcepsilonRI on mast cells inhibits B cell or mast cell activation. Activity of the inositol phosphatase SHIP is required for this negative signal. In vitro, SHIP catalyzes the conversion of the phosphoinositide 3-kinase (PI3K) product phosphatidylinositol 3,4, 5-trisphosphate (PIP3) into phosphatidylinositol 3,4-bisphosphate. Recent data demonstrate that coligation of FcgammaRIIb1 with BCR inhibits PIP3-dependent Btk (Bruton's tyrosine kinase) activation and the Btk-dependent generation of inositol trisphosphate that regulates sustained calcium influx. In this study, we provide evidence that coligation of FcgammaRIIb1 with BCR induces binding of PI3K to SHIP. This interaction is mediated by the binding of the SH2 domains of the p85 subunit of PI3K to a tyrosine-based motif in the C-terminal region of SHIP. Furthermore, the generation of phosphatidylinositol 3,4-bisphosphate was only partially reduced during coligation of BCR with FcgammaRIIb1 despite a drastic reduction in PIP3. In contrast to the complete inhibition of Tec kinase-dependent calcium signaling, activation of the serine/threonine kinase Akt was partially preserved during BCR and FcgammaRIIb1 coligation. The association of PI3K with SHIP may serve to activate PI3K and to regulate downstream events such as B cell activation-induced apoptosis.

3T3 Cells↗
PubMed1999

Impaired B cell development and proliferation in absence of phosphoinositide 3-kinase p85alpha.

Phosphoinositide 3-kinase (PI3K) activation has been implicated in many cellular responses, including fibroblast growth, transformation, survival, and chemotaxis. Although PI3K is activated by several agents that stimulate T and B cells, the role of PI3K in lymphocyte function is not clear. The mouse gene encoding the PI3K adapter subunit p85alpha and its splice variants p55alpha and p50alpha was disrupted. Most p85alpha-p55alpha-p50alpha-/- mice die within days after birth. Lymphocyte development and function was studied with the use of the RAG2-deficient blastocyst complementation system. Chimeric mice had reduced numbers of peripheral mature B cells and decreased serum immunoglobulin. The B cells that developed had diminished proliferative responses to antibody to immunoglobulin M, antibody to CD40, and lipopolysaccharide stimulation and decreased survival after incubation with interleukin-4. In contrast, T cell development and proliferation was normal. This phenotype is similar to defects observed in mice lacking the tyrosine kinase Btk.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1998

Phosphatidylinositol-3,4,5-trisphosphate (PtdIns-3,4,5-P3)/Tec kinase-dependent calcium signaling pathway: a target for SHIP-mediated inhibitory signals.

Tec family non-receptor tyrosine kinases have been implicated in signal transduction events initiated by cell surface receptors from a broad range of cell types, including an essential role in B-cell development. A unique feature of several Tec members among known tyrosine kinases is the presence of an N-terminal pleckstrin homology (PH) domain. We directly demonstrate that phosphatidylinositol-3,4,5-trisphosphate (PtdIns-3,4,5-P3) interacting with the PH domain acts as an upstream activation signal for Tec kinases, resulting in Tec kinase-dependent phospholipase Cgamma (PLCgamma) tyrosine phosphorylation and inositol trisphosphate production. In addition, we show that this pathway is blocked when an SH2-containing inositol phosphatase (SHIP)-dependent inhibitory receptor is engaged. Together, our results suggest a general mechanism whereby PtdIns-3,4,5-P3 regulates receptor-dependent calcium signals through the function of Tec kinases.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1998

Phosphoinositide kinases.

Phosphatidylinositol, a component of eukaryotic cell membranes, is unique among phospholipids in that its head group can be phosphorylated at multiple free hydroxyls. Several phosphorylated derivatives of phosphatidylinositol, collectively termed phosphoinositides, have been identified in eukaryotic cells from yeast to mammals. Phosphoinositides are involved in the regulation of diverse cellular processes, including proliferation, survival, cytoskeletal organization, vesicle trafficking, glucose transport, and platelet function. The enzymes that phosphorylate phosphatidylinositol and its derivatives are termed phosphoinositide kinases. Recent advances have challenged previous hypotheses about the substrate selectivity of different phosphoinositide kinase families. Here we re-examine the pathways of phosphoinositide synthesis and the enzymes involved.

1-Phosphatidylinositol 4-Kinase↗
PubMed1996

Competitive inhibition of calcineurin phosphatase activity by its autoinhibitory domain.

Calcineurin (protein phosphatase 2B), a calmodulin- and calcium-dependent serine/threonine phosphatase, appears to be regulated by a C-terminal autoinhibitory domain. A 25 amino acid peptide derived from this domain inhibits calcineurin phosphatase activity in vitro. Here we show that a 97 amino acid fragment of the calcineurin A alpha C-terminus is approx. 8-fold more potent than the shorter peptide in calcineurin inhibition experiments. Mutation of an evolutionarily conserved Asp to Asn, previously shown to disrupt calcium-dependent signalling and calcineurin regulation in T-lymphocytes, greatly reduced inhibition by the autoinhibitory domain in vitro. Kinetic analysis of wild-type and mutated autoinhibitory domains show that both are competitive inhibitors of calcineurin phosphatase activity with Ki values of 5.0 +/- 0.2 microM and 36.0 +/- 3.7 microM respectively. This suggests intrasteric regulation of calcineurin, with the autoinhibitory domains interacting at the active site of the enzyme. The competitive behaviour of the autoinhibitory domains contrasts with the mechanism of calcineurin inhibition by immunosuppressant-immunophilin complexes, which have been shown to bind to calcineurin at a region removed from the active site.

Allosteric Regulation↗
PubMed1996

Structural organization and alternative splicing of the murine phosphoinositide 3-kinase p85 alpha gene.

Phosphoinositide 3-kinase is a lipid and protein kinase composed of a 110-kDa catalytic subunit and an 85-kDa (p85) or 55-kDa (p55) regulatory subunit. In mammals, at least two genes encode catalytic subunits, and at least three genes encode regulatory subunits. Here we report the cloning and structural analysis of the mouse p85 alpha gene. The translated portion of mouse p85 alpha is encoded by 15 exons that span at least 40 kb. We have cloned an alternatively spliced form of p85 alpha from both mouse and rat cDNA libraries. This splice variant encodes a unique 5'-untranslated region, start codon, and 6-amino-acid aminoterminus followed by the carboxyterminal 418 amino acids of p85 alpha. A corresponding exon is present within the p85 alpha genomic locus. In vitro transcription and translation of the splice variant cDNA generate a protein of approximately 45 kDa that is reactive with an anti-p85 alpha antiserum. Northern blot analysis of mouse tissues reveals differential expression of full-length and alternatively spliced p85 alpha, with the splice variant most abundant in the liver.

Alternative Splicing↗
PubMed1996

Identification of a physical interaction between calcineurin and nuclear factor of activated T cells (NFATp).

In T lymphocytes, the calcium/calmodulin-dependent serine/threonine phosphatase, calcineurin, plays a pivotal role in transducing membrane-associated signals to the nucleus. One of the putative targets of calcineurin is the pre-existing, cytosolic component of the nuclear factor of activated T cells (NFATp; also referred to as NFAT1), which is one of several transcription factors required for the expression of interleukin 2. Inhibition of calcineurin by the immunosuppressive drugs cyclosporin A and FK506 prevents dephosphorylation of NFATp and its translocation to the nucleus. However, a physical interaction between calcineurin and NFATp has not been demonstrated. Here we demonstrate the binding of NFATp from lysates of T cells to immobilized calcineurin. Stimulation of T cells with calcium ionophore induced a shift in the molecular weight of NFATp that is due to its dephosphorylation. This dephosphorylation was inhibited by treatment of T cells with cyclosporin A or FK506 prior to stimulation. Of note, both the phosphorylated and the dephosphorylated form of NFATp bound to calcineurin. Furthermore, the binding of both forms of NFATp to calcineurin was inhibited by pretreatment of calcineurin with a complex of FK506 and its ligand FKBP12. Taken together these data strongly suggest a direct interaction of calcineurin with NFATp and that this interaction does not depend upon the phosphorylation sites of NFATp affected by activation.

Animals↗
PubMed1995

The complex of FK506-binding protein 12 and FK506 inhibits calcineurin phosphatase activity and IgE activation-induced cytokine transcripts, but not exocytosis, in mouse mast cells.

FK506 and cyclosporin A (CsA) are immunosuppressive agents that inhibit IL-2 production by activated T cells, but only CsA inhibits IgE activation-induced cytokine transcripts in mouse IL-3-dependent, bone marrow-derived mast cells (BMMC). We previously associated the resistance of BMMC to FK506 with a deficiency in the expression of FK506 binding protein (FKBP) 12, a molecule that forms a complex with FK506 capable of inhibiting calcineurin phosphatase activity in vitro. In this report, we establish that FKBP12 mediates FK506 inhibition of both calcineurin phosphatase activity and IgE activation-induced cytokine transcripts in a Kirsten murine sarcoma virus-immortalized mast cell line that is FKBP12 deficient. Overexpression of FKBP12 by transfection enhanced the ability of FK506 to inhibit calcineurin phosphatase activity (IC50 = 2 nM), compared with cells transfected with the expression vector alone (IC50 > 30 nM). The IC50 value for FK506 inhibition of IgE activation-induced transcripts for TNF-alpha decreased from 40 nM in vector control cells to 10 nM in FKBP12 transfectants. Similarly, the IC50 value for inhibition of IL-6 transcripts decreased from > 1000 nM in vector control cells to 35 nM in FKBP12 transfectants. In contrast, activation-elicited release of the secretory granule mediator beta-hexosaminidase was only partially inhibited by FK506 at 1000 nM, regardless of the levels of FKBP12 expressed by the cells. Thus, FKBP12 is the dominant cytosolic protein that mediates FK506 inhibition of TNF-alpha and IL-6 transcripts.

Animals↗