Class IA phosphoinositide 3-kinases (PI3Ks) are a family of p85/p110 heterodimeric lipid kinases that generate second messenger signals downstream of tyrosine kinases, thereby controlling cell metabolism, growth, proliferation, differentiation, motility, and survival. Mammals express three class IA catalytic subunits: p110α, p110β, and p110δ. It is unclear to what extent these p110 isoforms have overlapping or distinct biological roles. Mice expressing a catalytically inactive form of p110δ (p110δ D910A ) were generated by gene targeting. Antigen receptor signaling in B and T cells was impaired and immune responses in vivo were attenuated in p110δ mutant mice. They also developed inflammatory bowel disease. These results reveal a selective role for p110δ in immunity.

Treatment of human prostate carcinoma-derived LNCaP cells with androgen or oestradiol triggers simultaneous association of androgen receptor and oestradiol receptor b with Src, activates the Src/Raf-1/ Erk-2 pathway and stimulates cell proliferation. Surprisingly, either androgen or oestradiol action on each of these steps is inhibited by both anti-androgens and anti-oestrogens. Similar ®ndings for oestradiol receptor a were observed in MCF-7 or T47D cells stimulated by either oestradiol or androgens. Microinjection of LNCaP, MCF-7 and T47D cells with SrcK ± abolishes steroid-stimulated S-phase entry. Data from transfected Cos cells con®rm and extend the ®ndings from these cells. Hormone-stimulated Src interaction with the androgen receptor and oestradiol receptor a or b is detected using glutathione S-transferase fusion constructs. Src SH2 interacts with phosphotyrosine 537 of oestradiol receptor a and the Src SH3 domain with a proline-rich stretch of the androgen receptor. The role of this phosphotyrosine is stressed by its requirement for association of oestradiol receptor a with Src and consequent activation of Src in intact Cos cells.

The molecular mechanisms by which ovarian hormones stimulate growth of breast tumors are unclear. It has been reported previously that estrogens activate the signal-transducing Src/p21 ras /Erk pathway in human breast cancer cells via an interaction of estrogen receptor (ER) with c-Src. We now show that progestins stimulate human breast cancer T47D cell proliferation and induce a similar rapid and transient activation of the pathway which, surprisingly, is blocked not only by anti-progestins but also by anti-estrogens. In Cos-7 cells transfected with the B isoform of progesterone receptor (PR B ), progestin activation of the MAP kinase pathway depends on co-transfection of ER. A transcriptionally inactive PR B mutant also activates the signaling pathway, demonstrating that this activity is independent of transcriptional effects. PR B does not interact with c-Src but associates via the N-terminal 168 amino acids with ER. This association is required for the signaling pathway activation by progestins. We propose that ER transmits to the Src/p21 ras /Erk pathway signals received from the agonist-activated PR B . These findings reveal a hitherto unrecognized cross-talk between ovarian hormones which could be crucial for their growth-promoting effects on cancer cells.

No abstract

No abstract

The p110 isoforms of phosphoinositide 3-kinase (PI3K) are acutely regulated by extracellular stimuli. The class IA PI3K catalytic subunits (p110␣, p110␤, and p110␦) occur in complex with a Src homology 2 (SH2) domain-containing p85 regulatory subunit, which has been shown to link p110␣ and p110␦ to Tyr kinase signaling pathways. The p84/p101 regulatory subunits of the p110␥ class IB PI3K lack SH2 domains and instead couple p110␥ to G protein-coupled receptors (GPCRs). Here, we show, using smallmolecule inhibitors with selectivity for p110␤ and cells derived from a p110␤-deficient mouse line, that p110␤ is not a major effector of Tyr kinase signaling but couples to GPCRs. In macrophages, both p110␤ and p110␥ contributed to Akt activation induced by the GPCR agonist complement 5a, but not by the Tyr kinase ligand colony-stimulating factor-1. In fibroblasts, which express p110␤ but not p110␥, p110␤ mediated Akt activation by the GPCR ligands stromal cell-derived factor, sphingosine-1-phosphate, and lysophosphatidic acid but not by the Tyr kinase ligands PDGF, insulin, and insulin-like growth factor 1. Introduction of p110␥ in these cells reduced the contribution of p110␤ to GPCR signaling. Taken together, these data show that p110␤ and p110␥ can couple redundantly to the same GPCR agonists. p110␤, which shows a much broader tissue distribution than the leukocyterestricted p110␥, could thus provide a conduit for GPCR-linked PI3K signaling in the many cell types where p110␥ expression is low or absent.gene targeting ͉ signaling ͉ tyrosine kinase ͉ Akt ͉ insulin T he lipid second messengers generated by phosphoinositide 3-kinases (PI3Ks) regulate a wide variety of cellular functions such as cell growth, proliferation, differentiation, and survival and have been implicated in cancer, inflammation, and diabetes. Mammals have eight isoforms of PI3K, which have been divided in three classes (1). Thus far, attention has focused mainly on the class I PI3Ks that are acutely activated by extracellular ligands. These heterodimers consist of a p110 catalytic subunit in complex with a regulatory subunit and have further been subdivided into class IA and IB PI3Ks. The class IA catalytic subunits (p110␣, p110␤, and p110␦) are in complex with an Src homology 2 (SH2) domain-containing regulatory subunit (of which there are five species, often referred to as p85s) that binds phosphoTyr in intracellular proteins, allowing recruitment of p85/p110 complexes to the membrane. The class IB regulatory subunits (p84, p101) do not have SH2 domains and link the single class IB PI3K catalytic subunit (p110␥) to G protein-coupled receptors (GPCRs).Over the last few years, the cellular signaling contexts and physiological roles of p110␣, p110␦, and p110␥ have become clearer, because of the generation of gene-targeted mice and small-molecule inhibitors for these PI3K isoforms. In contrast, very little is known about p110␤, both at the cellular and organismal level. One confounding factor has been the very early embryonic lethality of the p110␤ KO ...

No abstract

The role of PI3K in T cell activation and costimulation has been controversial. We previously reported that a kinase-inactivating mutation (D910A) in the p110δ isoform of PI3K results in normal T cell development, but impaired TCR-stimulated cell proliferation in vitro. This proliferative defect can be overcome by providing CD28 costimulation, which raises the question as to whether p110δ activity plays a role in T cell activation in vivo, which occurs primarily in the context of costimulation. In this study, we show that the PI3K signaling pathway in CD28-costimulated p110δD910A/D910A T cells is impaired, but that ERK phosphorylation and NF-κB nuclear translocation are unaffected. Under in vitro conditions of physiological Ag presentation and costimulation, p110δD910A/D910A T cells showed normal survival, but underwent fewer divisions. Differentiation along the Th1 and Th2 lineages was impaired in p110δD910A/D910A T cells and could not be rescued by exogenous cytokines in vitro. Adoptive transfer and immunization experiments in mice revealed that clonal expansion and differentiation in response to Ag and physiological costimulation were also compromised. Thus, p110δ contributes significantly to Th cell expansion and differentiation in vitro and in vivo, also in the context of CD28 costimulation.