Costimulation of resting B lymphocytes alters the IL－4－activated IRS2 signaling pathway in a STAT6 independent manner：implications for cell survival and proliferation

IL-4 is an important B cell survival and growth factor. IL-4 induced the tyrosine phosphorylation of IRS2 in resting B lymphocytes and in LPS- or CD40L-activated blasts. Phosphorylated IRS2 coprecipitated with the p85 subunit of PI 3' kinase in both resting and activated cells. By contrast, association of phosphorylated IRS2 with GRB2 was not detected in resting B cells after IL-4 treatment although both proteins were expressed. However, IL-4 induced association of IRS2 with GRB2 in B cell blasts. The pattern of IL-4-induced recruitment of p85 and GRB2 to IRS2 observed in B cells derived from STAT6 null mice was identical to that observed for normal mice. While IL-4 alone does not induce activation of MEK, a MEK1 inhibitor suppressed the IL-4-induced proliferative response of LPS-activated B cell blasts. These results demonstrate that costimulation of splenic B cells alters IL-4-induced signal transduction independent of STAT6 leading to proliferation. Furthermore, proliferation induced by IL-4 in LPS-activated blasts is dependent upon the MAP kinase pathway.     

The kinetics of IL—4 and IFN—γ gene expression in Mice after Trichosansin immunization

Trichosanthin (TCS) is a potent allergen to mice.According to our previous experiments,it could bring out the IgE response to ovabumin (OVA) if TCS was given one day before OVA immunization,while OVA alone could not induce IgE to it.In this work,the kinetics of interleukin 4(IL-4) and interferon γ(IFN-γ) gene expression in the mesenteric lymph node (MLN) of TCS-immunized mice was investigated using a semi-quantitative RT-PCR method.It indicated that TCS induced significant IL-4 gene expression and the peaks of IL4 gene expression were on day one after TCS immunization in both primary and secondary response.In contrast,the IFN-γ gene expression was suppressed.Furthermor,the IL-4 gene expression in the secondary response was lower than that in the primary response.Thus the presence of IgE memory B cells were studied.Results showed that the amount of mature IgE mRNA arose significantly and rapidly one day after TCS restimulation,while in the MLN of the mice primed 30 days before and without boost,it was almost as the same amount of the unimmunized control.These findings suggest the existence of the IgE memory B cells in the mice after the primary TCS immunization.     

The magic of four： induction of pluripotent stem cells from somatic cells by Oct4, Sox2, Myc and Klf4

The developmental process from a fertilized egg to agrown adult is programmed with remarkable accuracy.While the genetic information of the fertilized egg and itsdescendent somatic cells are the same, it is the selectiveexpressions of the same genome that give rise to the 200or so different cell types in an adult. The differentiatedstates of these adult cells are maintained epigenetically,presumably through the modification of chromatins and theassociated histones. In higher mammals, it was thought thatthe differentiation process is irreversible until the successfulcloning of Dolly [1]. By transferring a nucleus from a fullydifferentiated cell in the mammary gland, Wilmut and col-leagues were able to generate an exact replica of a highermammal, the Doily [1]. This work not only demonstratedthat the genome of differentiated cells can be reprogrammedinto an embryonic state and then to resume a full-fledgeddevelopmental process to generate a normal adult, but alsorejuvenated the field of animal cloning. The prospect thata somatic cell from a patient may be reprogrammed to the     

Signal transduction mediated by Bid,a pro—death Bcl—2 family proteins,connects the death receptor and mitochondria apoptosis pathways

Two major apoptosis pathways have been defined in mammalian cells,the Fas/TNF-R1 death receptor pathway and the mitochondria pathway.The Bcl-2 family proteins consist of both anti-apoptosis and pro-apoptosis members that regulate apoptosis,mainly by controlling the release of cytochrome c and other mitochondrial apoptotic events.However,death signals mediated by Fas/TNF-R1 receptors can usually activate caspases directly,bypassing the need for mitochondria and escaping the regulation by Bcl-2 family proteins.Bid is a novel pro-apoptosis Bcl-2 family protein that is activated by caspase 8 in response to Fas/TNF-R1 death receptor signals.Activated Bid is translocated to mitochondria and induces cytochrome c release,which in turn activates downstream caspases.Such a connection between the two apoptosis pathways could be important for induction of apoptosis in certain types of cells and responsible for the pathogenesis of a number of human diseases.     

Spatial and temporal regulation of collagenases—3,—4,and stromelysin —3 implicates distinct functions in apoptosis and tissue remodeling during frog metamorphosis

Matrix metalloproteinases (MMPs) are a family of extracellular proteases capable of degrading various proteinaceous components of the extracellular matrix(ECM).They have been implicated to play important roles in a number of developmental and pathological processes,such as tumor metastasis and inflammation.Relatively few studies have been carried out to investigate the function of MMPs during postembryonic organ-development.Using Xenopus laevis development as a model system,we demonstrate here that three MMPs,stromelysin-3(ST3),collagenases-3(Col3),and Col4,have distinct spatial and temporal expression profiles during metamorphosis as the tadpole transforms into a frog.In situ hybridizations reveal a tight,but distinct,association of individual MMPs with tissue remodeling in the tail and intestine during metamorphosis.In particular,ST3 expression is strongly correlated with apoptosis in both organs as demonstrated by analyses of serial sections with in situ hybridization for ST3 mRNA and TUNEL (terminal deoxyribonucleotidyl transferase-mediated dUTP-biotin nick end labeling)for apoptosis,respectively.On the other hand,Col3 and Col4 are present in regions where extensive connective tissue remodeling take place.These results indicate that ST3 is likely to play a role in ECM-remodeling that facilitate apoptotic tissue remodeling or resorption while Col3 and Col4 appear to participate in connective tissue degradation during development.     

Regulation of cell migration and survival by focal adhesion targeting of Lasp-1

Large-scale proteomic and functional analysis of isolated pseudopodia revealed the Lim, actin, and SH3 domain protein (Lasp-1) as a novel protein necessary for cell migration, but not adhesion to, the extracellular matrix (ECM). Lasp-1 is a ubiquitously expressed actin-binding protein with a unique domain configuration containing SH3 and LIM domains, and is overexpressed in 8-12% of human breast cancers. We find that stimulation of nonmotile and quiescent cells with growth factors or ECM proteins facilitates Lasp-1 relocalization from the cell periphery to the leading edge of the pseudopodium, where it associates with nascent focal complexes and areas of actin polymerization. Interestingly, although Lasp-1 dynamics in migratory cells occur independently of c-Abl kinase activity and tyrosine phosphorylation, c-Abl activation by apoptotic agents specifically promotes phosphorylation of Lasp-1 at tyrosine 171, which is associated with the loss of Lasp-1 localization to focal adhesions and induction of cell death. Thus, Lasp-1 is a dynamic focal adhesion protein necessary for cell migration and survival in response to growth factors and ECM proteins.     

Regulation of expression and function of Lck tyrosine kinase by high cell density

For many types of cells, an increase in cell density leads to characteristic changes in intracellular signalling and cell function. It is unknown, however, whether cell density affects the function of T lymphocytes. It is presented here that aggregation of Jurkat T cells, murine thymocytes or human peripheral blood T cells, results in gradual modification of the Lck tyrosine kinase. Within one hour of aggregation, Lck in the detergent-insoluble lipid raft fraction is dephosphorylated mainly at the carboxy-terminal tyrosine. Further aggregation leads to gradual loss of Lck protein from both lipid raft and non-raft fractions which is accompanied by increased protein ubiquitination, a process that is more evident in the detergent-soluble fraction. In contrast, the expression of LAT, which like Lck distributes to raft and non-raft membrane, or Csk, a kinase with a structure similar to Lck, is not affected by cell aggregation. Dephosphorylation of lipid raft-associated Lck, albeit with reduced kinetics, is observed in aggregated Jurkat CD45-deficient cells as well, suggesting involvement of additional tyrosine phosphatases. Changes in Lck structure and expression correlate with reduced ability of aggregated cells to fully activate protein tyrosine phosphorylation after stimulation of the TCR, and with changes in the activation of down-stream signalling cascades.     

Receptor-like protein tyrosine phosphatases: alike and yet so different

Reversible phosphorylation on tyrosine residues is an extremely rapid and powerful posttranslational modification that is used in signalling pathways for the regulation of cell growth and differentiation. Over the past several years an impressive number of receptor-like protein tyrosine phosphatase (RPTPase) family members have been identified by molecular cloning, and undoubtedly many more will follow. This review provides an overview of the molecular data that are available for the currently identified RPTPases and discusses their possible biological implications. This revised version was published online in June 2006 with corrections to the Cover Date.     

Multiple signaling pathways in bovine chromaffin cells regulate tyrosine hydroxylase phosphorylation at Ser19, Ser31, and Ser40

Intact bovine adrenal medullary chromaffin cells were preincubated with³²PO₄, and the multiplesite phosphorylation of tyrosine hydroxylase (TH) was studied. Up to eight³²P-labeled peptides were produced by tryptic hydrolysis of TH; however, all of the tryptic phosphopeptides were derived from four phosphorylation sites—Ser⁸, Ser¹⁹, Ser³¹ and Ser⁴⁰. In situ regulation of³²P incorporation into the latter three sites was demonstrated with a diverse set of pharmacological agents.³²P incorporation into Ser¹⁹ was preferentially increased by brief exposures to depolarizing secretagogues. Longer treatments also increased Ser³¹ and Ser⁴⁰ phosphorylation. Nicotine, muscarine and vasoactive intestinal polypeptide—reflecting cholinergic and non-cholinergic components of sympatho-adrenal transmission—each produced different patterns of multiple-site phosphorylation of TH. Nicotine, bradykinin and histamine increased³²P incorporation at each of the three sites whereas muscarine, angiotensin II, endothelin III, prostaglandin E1, GABA and ATP selectively increased Ser³¹ phosphorylation. Nerve growth factor did not influence TH phosphorylation in chromaffin cells from adult adrenal glands but selectively increased Ser³¹ phosphorylation in chromaffin cells isolated from calf adrenal glands.³²P incorporation into Ser⁴⁰ was selectively increased by forskolin and other cAMP-acting agents whereas vasoactive intestinal polypeptide increased Ser³¹ and Ser⁴⁰ phosphorylation. Thus, the phosphorylation of TH in bovine chromaffin cells appears to be regulated at three sites by three separate intracellular signaling pathways—Ser¹⁹ via Ca²⁺/calmodulin-dependent protein kinase II; Ser³¹ via ERK (MAP2 kinases); and Ser⁴⁰ via cAMP-dependent protein kinase. These signaling pathways, as well as the extracellular signals that were effective in stimulating them, are similar to those previously described for TH in rat pheochromocytoma cells. However, several of the pharmacological agents produced different patterns of multiple-site TH phosphorylation in the bovine chromaffin cells. These differences between tissues could be accounted for by differences in the coupling/access between the extracellular signal transduction systems and the intracellular signaling pathways as opposed to differences in the intracellular signaling pathwaysper se.Special issue dedicated to Dr. Paul Greengard     

Suppression of Pyk2 kinase and cellular activities by FIP200

Proline-rich tyrosine kinase 2 (Pyk2) is a cytoplasmic tyrosine kinase implicated to play a role in several intracellular signaling pathways. We report the identification of a novel Pyk2-interacting protein designated FIP200 (FAK family kinase-interacting protein of 200 kD) by using a yeast two-hybrid screen. In vitro binding assays and coimmunoprecipitation confirmed association of FIP200 with Pyk2, and similar assays also showed FIP200 binding to FAK. However, immunofluorescent staining indicated that FIP200 was predominantly localized in the cytoplasm. FIP200 bound to the kinase domain of Pyk2 and inhibited its kinase activity in in vitro kinase assays. FIP200 also inhibited the kinase activity of the Pyk2 isolated from SYF cells (deficient in Src, Yes, and Fyn expression) and the Pyk2 mutant lacking binding site for Src, suggesting that it regulated Pyk2 kinase directly rather than affecting the associated Src family kinases. Consistent with its inhibitory effect in vitro, FIP200 inhibited activation of Pyk2 and Pyk2-induced apoptosis in intact cells, which correlated with its binding to Pyk2. Finally, activation of Pyk2 by several biological stimuli correlated with the dissociation of endogenous FIP200-Pyk2 complex, which provided further support for inhibition of Pyk2 by FIP200 in intact cells. Together, these results suggest that FIP200 functions as an inhibitor of Pyk2 via binding to its kinase domain.