SOCS proteins: negative regulators of cytokine signaling.

Cytokines regulate the growth and differentiation of cells by binding to cell-surface receptors and activating intracellular signal transduction cascades such as the JAK-STAT pathway. Cytokine signaling is negatively regulated with respect to both magnitude and duration, and it is now clear that the suppressor of cytokine signaling (SOCS) family of proteins (SOCS1-SOCS7 and CIS) contributes significantly to this process. Transcripts encoding CIS, SOCS1, SOCS2, and SOCS3 are upregulated in response to cytokine stimulation, and the corresponding SOCS proteins inhibit cytokine-induced signaling pathways. SOCS proteins therefore form part of a classical negative feedback circuit. SOCS family members modulate signaling by several mechanisms, which include inactivation of the Janus kinases (JAKs), blocking access of the signal transducers and activators of transcription (STATs) to receptor binding sites, and ubiquitination of signaling proteins and their subsequent targeting to the proteasome. Gene targeting has been used to generate mice lacking socs1, socs2, or socs3, in order to elucidate the physiological function of these SOCS family members. The analysis of socs1(-/-) mice has revealed that SOCS1 plays a key role in the negative regulation of interferon-gamma signaling and in T cell differentiation. Socs2(-/-) mice are 30%-40% larger than wild-type mice, demonstrating that SOCS2 is a critical regulator of postnatal growth. Additionally, the study of embryos lacking socs3 has revealed that SOCS3 is an important regulator of fetal liver hematopoiesis. The biological role of other SOCS proteins remains to be determined.

SOCS: physiological suppressors of cytokine signaling.

Cytokines regulate cellular behavior by interacting with receptors on the plasma membrane of target cells and activating intracellular signal transduction cascades such as the JAK-STAT pathway. Suppressors of cytokine signaling (SOCS) proteins negatively regulate cytokine signaling. The SOCS family consists of eight proteins: SOCS1-SOCS7 and CIS, each of which contains a central Src-homology 2 (SH2) domain and a C-terminal SOCS box. The expression of CIS, SOCS1, SOCS2 and SOCS3 is induced in response to stimulation by a wide variety of cytokines, and overexpression of these proteins in cell lines results in inhibition of cytokine signaling. Thus, SOCS proteins appear to form part of a classical negative feedback loop. The analysis of mice lacking SOCS1 has revealed that it is critical in the negative regulation of IFN(gamma) signaling and in the differentiation of T cells. Additionally, the analysis of mouse embryos lacking SOCS3 suggests that SOCS3 negatively regulates fetal liver erythropoiesis, probably through its ability to modulate erythropoietin (Epo) signaling. Thus, the use of gene targeting has confirmed that SOCS proteins regulate cytokine signaling in a physiological setting.

The evolution of moral dispositions in the human species.

Kohlberg's model of moral development is viewed from the perspective of evolutionary biology. Moral judgments defining Kohlberg's stages of moral development are seen as manifestations of structures evolved to uphold systems of cooperation. Game theory research on adaptive strategies of cooperation supports the conclusion that humans inherit dispositions to uphold the systems of cooperation implicit in the first three stages in Kohlberg's sequence, but not the systems of cooperation implicit in the highest stages. The empirical evidence on real-life morality is more consistent with a biological model of ontogenesis than is the model espoused by Kohlbergians. Although people occasionally make moral judgments in their everyday lives to reveal their solutions to moral dilemmas, as Kohlberg's model assumes, they more often make moral decisions that advance their adaptive interests.

The B cell antigen receptor activates the Akt (protein kinase B)/glycogen synthase kinase-3 signaling pathway via phosphatidylinositol 3-kinase.

We have previously shown that the B cell Ag receptor (BCR) activates phosphatidylinositol (PI) 3-kinase. We now show that a serine/threonine kinase called Akt or protein kinase B is a downstream target of PI 3-kinase in B cells. Akt has been shown to promote cell survival as well as the transcription and translation of proteins involved in cell cycle progression. Using an Ab that specifically recognizes the activated form of Akt that is phosphorylated on serine 473, we show that BCR engagement activates Akt in a PI 3-kinase-dependent manner. These results were confirmed using in vitro kinase assays. Moreover, BCR ligation also induced phosphorylation of Akt of threonine 308, another modification that is required for activation of Akt. In the DT40 chicken B cell line, phosphorylation of Akt on serine 473 was completely dependent on the Lyn tyrosine kinase, while the Syk tyrosine kinase was required for sustained phosphorylation of Akt. Complementary experiments in BCR-expressing AtT20 endocrine cells confirmed that Src kinases are sufficient for BCR-induced Akt phosphorylation, but that Syk is required for sustained phosphorylation of Akt on both serine 473 and threonine 308. In insulin-responsive cells, Akt phosphorylates and inactivates the serine/threonine kinase glycogen synthase kinase-3 (GSK-3). Inactivation of GSK-3 may promote nuclear accumulation of several transcription factors, including NF-ATc. We found that BCR engagement induced GSK-3 phosphorylation and decreased GSK-3 enzyme activity. Thus, BCR ligation initiates a PI 3-kinase/Akt/GSK-3 signaling pathway.

An 11-amino acid sequence in the cytoplasmic domain of CD40 is sufficient for activation of c-Jun N-terminal kinase, activation of MAPKAP kinase-2, phosphorylation of I kappa B alpha, and protection of WEHI-231 cells from anti-IgM-induced growth arrest.

We have previously shown that CD40 causes strong activation of the c-Jun N-terminal kinase (JNK), the p38 mitogen-activated protein kinases (MAPK) and MAPKAP kinase-2, a downstream target of p38 MAPK. To identify signaling motifs in the CD40 cytoplasmic domain that are responsible for activation of these kinases, we have created a set of 11 chimeric receptors consisting of the extracellular and transmembrane domains of CD8 fused to portions of the murine CD40 cytoplasmic domain. These chimeric receptors were expressed in WEHI-231 B lymphoma cells. We found that amino acids 35-45 of the CD40 cytoplasmic domain constitute an independent signaling motif that is sufficient for activation of the JNK and p38 MAPK pathways, as well as for induction of I kappa B alpha phosphorylation and degradation. Amino acids 35-45 were also sufficient to protect WEHI-231 cells from anti-IgM-induced growth arrest. This is the same region of CD40 required for binding the TNF receptor-associated factor-2 (TRAF2), TRAF3, and TRAF5 adapter proteins. These data support the idea that one or more of these TRAF proteins couple CD40 to the kinase cascades that activate NF-kappa B, JNK, and p38 MAPK.

Rapid and efficient retrovirus-mediated gene transfer into B cell lines.

Murine B cell lines such as WEHI-231, BAL17 and M12.4.1 are frequently used as model systems to study signal transduction, cell cycle regulation, and apoptosis. Dissection of these processes often involves expressing exogenous genes in these cells. Electroporation is an inefficient method to express genes in B cell lines and requires several weeks to isolate and analyze clones, followed by an additional one to two weeks to grow sufficient cells for biochemical experiments (e.g. immunoprecipitations). In this report, we describe an optimized procedure for retroviral-mediated gene transfer into murine B cell lines that allows one to obtain a pure population of cells expressing an exogenous gene within 4 days. Two days post-infection, between 10% (BAL17 and M12.4.1 cells) and 70% (WEHI-231 cells) of the cells express the exogenous gene. Culturing the cells for an additional 48 hours with puromycin kills all the non-infected cells and yields a pure population of cells that express the exogenous gene. Sufficient cells for biochemical experiments can be obtained by expanding the cell culture for an additional 5 to 7 days. This rapid and efficient retroviral-mediated gene transfer procedure can greatly expedite the study of signal transduction and other processes in B cells.

B cell antigen receptor signaling induces the formation of complexes containing the Crk adapter proteins.

Crk proteins are Src homology (SH) 2/SH3-containing adapter proteins that can mediate the formation of signaling complexes. We show that engaging the B cell antigen receptor (BCR) on the RAMOS B cell line caused both Crk-L and Crk II to associate with several tyrosine-phosphorylated proteins. We identified two of these phosphoproteins as Cas and Cbl and showed that both bound to the Crk SH2 domain after BCR engagement. BCR ligation also increased the amount of Crk proteins in the particulate fraction of the cells and induced the formation of Crk.Cas and Crk.Cbl complexes in the particulate fraction. We propose that tyrosine phosphorylation of membrane-associated Cas and Cbl creates binding sites for the Crk SH2 domain and recruits Crk complexes to cellular membranes. Thus, Crk proteins may participate in BCR signaling by using their SH2 domains to direct the interactions and subcellular localization of proteins that bind to their SH3 domains. In RAMOS cells, we found that the SH3 domains of Crk-L and Crk II bound C3G. Since C3G activates Rap, a negative regulator of the Ras pathway, Crk proteins may participate in regulation of Ras signaling by the BCR.

Evaluation of two-dimensional phosphopeptide maps by electrospray ionization mass spectrometry of recovered peptides.

Intracellular signaling pathways are to a large extent regulated by reversible protein phosphorylation of pathway components. To fully investigate the regulation of these pathways, it is often necessary to identify the sites of protein phosphorylation induced on individual components. The low abundance of many of these molecules and the potentially low stoichiometry of phosphorylation means that conventional analytical techniques are incapable of identifying specific sites of modification inducible in vivo. The most common technique used is two-dimensional (2D) phosphopeptide mapping (electrophoresis, thin-layer chromatography) of peptides derived by proteolysis of a phosphoprotein. The number of spots detected is commonly interpreted as the number of sites of phosphorylation. Here we have achieved positive identification of phosphorylation sites by capillary high-performance liquid chromatography, with on-line mass spectrometric detection, of phosphopeptides recovered from 2D phosphopeptide maps. We demonstrate that the chemical composition of phosphopeptides is not altered during the 2D mapping procedure. By detailed analysis of the sites of phosphorylation induced in vitro on CD3-zeta by p56lck we demonstrate that interpretation of the sites of phosphorylation based on 2D phosphopeptide mapping alone is difficult. To minimize over- or misinterpretation of 2D phosphopeptide maps we therefore postulate rules that should be applied generally in cases in which protein phosphorylation sites are being evaluated by 2D phosphopeptide patterns alone.

Identification by electrospray ionization mass spectrometry of the sites of tyrosine phosphorylation induced in activated Jurkat T cells on the protein tyrosine kinase ZAP-70.

We have developed a rapid and sensitive two capillary-column chromatography and mass spectrometry-based method for the determination of protein phosphorylation sites following recovery of individual phosphopeptides from two-dimensional phosphopeptide maps. With a standard phosphopeptide, we demonstrate detection sensitivity of at least 250 fmol for this system. We applied this technique to the analysis of in vitro sites of tyrosine phosphorylation induced on the T cell-specific protein tyrosine kinase ZAP-70 in the absence and presence of p56lck. We show that ZAP-70 has a primary autophosphorylation site at Tyr-292, with a secondary site at Tyr-126. We also show additional phosphorylation at Tyr-69, Tyr-178, Tyr-492, and Tyr-493 upon the addition of the protein tyrosine kinase, p56lck. By comparative two-dimensional phosphopeptide mapping, we show that ZAP-70 isolated from Jurkat T cells also autophosphorylates at Tyr-292 and Tyr-126. Similar analysis of 32P-labeled Jurkat cells stimulated with anti-T cell receptor antibodies reveals Tyr-492 and Tyr-493 as the principal sites of T cell antigen receptor-induced tyrosine phosphorylation, with additional phosphorylation at the Tyr-292, but not the Tyr-126 autophosphorylation site. The high degree of sensitivity achieved with this technology should greatly facilitate the direct biochemical determination of inducible protein phosphorylation events, an experimental strategy that until now has been both time consuming and difficult.

The structural consistency of moral judgments about AIDS.

This study compared the structure (i.e., stage) of moral judgments to dilemmas involving AIDS to the structure of moral judgment on Kohlberg's test to determine whether attitudes and opinions about AIDS affect level of moral judgment. Subjects included 40 men, who responded to (a) two standard dilemmas from Colby and Kohlberg's (1987) test of moral judgment, (b) two of Kohlberg's dilemmas ammended to involve AIDS, (c) a set of AIDS dilemmas derived from incidents reported in the media, (d) a set of questions tapping attitudes toward people with AIDS, and (e) Rubin and Peplau's (1975) just-world scale. Moral judgment was structurally consistent across the three sets of dilemmas, and only weakly related to attitudes towards AIDS and belief in a just world. Moral choices did not covary with moral stage. These results are consistent with Kohlberg's contention that moral judgment is organized in "structures of the whole."

Coping, defending, and the relations between moral judgment and moral behavior in prostitutes and other female juvenile delinquents.

Twenty female juvenile delinquents who acknowledged engaging in prostitution, 20 juvenile delinquents who denied doing so, and 20 same-age control subjects responded to Colby and Kohlberg's (1987) Moral Judgment Interview (MJI), a moral dilemma about prostitution, and Joffe and Naditch's (1977) test of coping and defending. Delinquents scored lower on moral maturity and coping and higher on defensiveness than nondelinquents. Post hoc analyses revealed that low-coping delinquents (but not high-coping delinquents or control subjects) made significantly lower level moral judgments on the prostitution dilemma than on the less personally relevant MJI dilemmas. Groups did not differ in their prescriptive judgments on the MJI, but prostitutes made weaker judgments against prostitution than the other delinquents. Prescriptive judgments were not related to moral maturity. The results elucidate the relation between moral judgment and moral behavior.

Situational variation in moral judgment: In a stage or on a stage?

Two issues were examined in this study-the consistency of moral judgment across different types of dilemma and different social contexts, and the relationship between the structure (stage) of moral judgment and the content of moral decisions. Forty subjects were given two hypothetical dilemmas about business decisions and two standard Kohlberg dilemmas. Half the subjects directed their responses to a business audience, half to a philosophical audience. Responses to the moral dilemmas were scored in accordance with the Colby and Kohlberg (1987) scoring manual. Stage of moral reasoning was found to be significantly higher on the Kohlberg dilemmas than on the business dilemmas. A significant interaction between type of dilemma and audience was attributed to the tendency of subjects directing their responses to a business audience to interpret one of the business dilemmas in terms of the moral order of business, but for subjects directing their responses to a philosophy audience to treat it as a philosophical dilemma. The other business dilemma evoked uniformly low-level moral judgments. The amount of selfishness intrinsic in subjects' moral choices on the business dilemmas was significantly negatively correlated with moral maturity on the business dilemmas, but not with their moral maturity on Kohlberg's test. These results are interpreted as more consistent with models of moral development such as those advanced by C. G. Levine ([1979] "Stage Acquisition and Stage Use: An Appraisal of Stage Displacement Explanations of Variation in Moral Reasoning, " Human Development, Vol. 22, pp. 145-164), J. Rest ([1983] "Morality," in: P. H. Mussen [ed.], J. H. Flavell and E. Markman [Vol. eds.], Handbook of Child Psychology [Vol. 3, 4th ed.], John Wiley & Sons, New York), and R. Harré ([1984]) Personal Being: A Theory for Individual Psychology, Harvard University Press, Cambridge, Massachusetts), which posit a relatively wide range of within-person stage use and emphasize the determining power of social situations, than with the more constructivistic model of moral development of Colby and Kohlberg (1987).

Structural flexibility of moral judgment.

One of the central assumptions of Kohlberg's theory of moral development--that moral judgment is organized in structures of the whole--was examined. Thirty men and 30 women were given 2 dilemmas from Kohlberg's Moral Judgment Interview, a 3rd involving prosocial behavior, and a 4th involving impaired driving. Half the Ss responded to the prosocial and impaired-driving dilemmas from the perspective of a hypothetical character, and half responded from the perspective of the self. No sex or perspective differences in moral maturity were observed. Ss scored highest in moral maturity on Kohlberg's dilemmas, intermediate on the prosocial dilemma, and lowest on the impaired-driving dilemma. In partial support of Kohlberg's contention that his test assesses moral competence, there was a negative linear relationship between scores on his test and the proportion of Stage 2 judgments on the 2 other dilemmas. An interactional model of moral judgment is advanced.