Targeting of IL-2 to cytotoxic lymphocytes as an improved method of cytokine-driven immunotherapy

The use of high-dose interleukin-2 (IL-2) has fallen out of favor due to severe life-threatening side effects. We have recently described a unique way of directly targeting IL-2 to cytotoxic lymphocytes using a virally encoded immune evasion protein and an IL-2 mutant that avoids off-target side effects such as activation of regulatory T cells and vascular endothelium

p120 GAP Modulates Ras Activation of Jun Kinases and Transformation

Although recent evidence demonstrates that Ras causes transformation by activation of multiple downstream pathways, the specific role of non-Raf effector pathways is presently unknown. Although Ras causes activation of the Jun NH2-terminal kinases (JNKs) via a Raf-independent pathway, the contribution of JNK activation to Ras transformation and the effector that mediates JNK activation have not been established. We observed that a dominant negative mutant of SEK1/JNKK, an activator of JNKs, selectively inhibited oncogenic Ras activation of JNK and Ras transformation, but not Ras activation of the p42 mitogen-activated protein kinase. In contrast, overexpression of wild type SEK1 enhanced Ras activation of JNK and transforming activity. Thus, JNK activation promotes Ras transformation. Furthermore, a dominant negative mutant of p120 GAP (designated N-GAP), a candidate Ras effector, blocked Ras, but not Raf, transformation and blocked Ras, but not Rac, activation of JNK. Since N-GAP overexpression reduced the association of p190 Rac/Rho GAP with endogenous p120 GAP, N-GAP may form nonproductive complexes with components critical for p120 GAP function. In summary, p120 GAP may function as an effector for Ras activation of JNK and Ras transformation

Measuring Essential Learning Outcomes for Public Speaking

Basic Course Directors (BCDs) are typically expected to assess course learning outcomes, but few formal guidelines and resources exist for new BCDs. As one part of a larger multi-methodological assessment tool development project, this manuscript maps existing quantitative measures onto the six essential competencies and associated learning outcomes established by the Social Science Research Council Panel on Public Speaking. This manuscript compiles dozens of measurement resources, aligned by outcome, and also identifies areas where future assessment measures development is needed. While there are many measures available for evaluating outcomes related to creating messages, critically analyzing messages, and demonstrating self-efficacy, there are measurement gaps for outcomes related to communication ethics, embracing difference, and influencing public discourse

SMAD and p38 MAPK Signaling Pathways Independently Regulate α1(I) Collagen Gene Expression in Unstimulated and Transforming Growth Factor-β-stimulated Hepatic Stellate Cells

The hepatic stellate cell (HSC) is the predominant cell type responsible for excess collagen deposition during liver fibrosis. Both transforming growth factor-beta (TGF-beta), the most potent fibrogenic cytokine for HSCs, which classically activates Smad signaling, and p38 MAPK signaling have been shown to influence collagen gene expression; however, the relative contribution and mechanisms that these two signaling pathways have in regulating collagen gene expression have not been investigated. The aim of this study was to investigate the relative roles and mechanisms of both Smad and p38 MAPK signaling in alpha1(I) collagen gene expression in HSCs. Inhibiting either p38 MAPK or Smad signaling reduced alpha1(I) collagen mRNA expression in untreated or TGF-beta-treated HSCs, and when both signaling pathways were simultaneously inhibited, alpha1(I) collagen gene expression was essentially blocked. Both signaling pathways were found to independently and additively increase alpha1(I) collagen gene expression by transcriptional mechanisms. TGF-beta treatment increased alpha1(I) collagen mRNA half-life, mediated by increased stability of alpha1(I) collagen mRNA through p38 MAPK signaling but not through Smad signaling. In conclusion, both p38 MAPK and Smad signaling independently and additively regulate alpha1(I) collagen gene expression by transcriptional activation, whereas p38 MAPK and not Smad signaling increased alpha1(I) collagen mRNA stability

Tumor necrosis factor alpha stimulates AP-1 activity through prolonged activation of the c-Jun kinase.

Tumor necrosis factor alpha (TNF alpha) has multiple biological functions including the prolonged activation of the collagenase and c-jun genes, which are regulated via their AP-1 binding sites. We show that incubating human fibroblasts with TNF alpha induces prolonged activation of JNK, the c-Jun kinase, which phosphorylates the transactivation domain of c-Jun. Furthermore, an immune complex kinase assay specifically demonstrates that TNF alpha stimulates the activity of JNK1, the recently described predominant form of JNK. TNF alpha also produces a small and transient increase in extracellular signal-regulated kinase (ERK) activity and no measured increase in Raf-1 kinase activity. On the other hand, epidermal growth factor causes a prolonged activation of Raf-1 kinase and ERK activity and a smaller, more transient activation of JNK, whereas the phorbol ester phorbol 12-myristate 13-acetate causes a small stimulation of Raf-1 kinase and a pronounced stimulation of ERK activity. The activation of JNK by TNF alpha does not correlate with Raf-1 or ERK activity. The kinetics of Raf-1, ERK, and JNK induction by epidermal growth factor, phorbol 12-myristate 13-acetate, or TNF alpha indicate distinct mechanisms of activation in human fibroblasts

Physical Association of PDK1 with AKT1 Is Sufficient for Pathway Activation Independent of Membrane Localization and Phosphatidylinositol 3 Kinase

Frequent activation of the AKT serine-threonine kinase in cancer confers resistance to therapy. AKT is activated by a multi-step process involving phosphatidylinositide (PtdIns) phosphate-mediated recruitment of AKT and its upstream kinases, including 3-Phosphoinositide-dependent kinase 1 (PDK1), to the inner surface of the cell membrane. PDK1 in the appropriate context phosphorylates AKT at threonine 308 (T308) to activate AKT. Whether PtdIns(3,4,5)Ps (PtdInsP3) binding and AKT membrane translocation mediate functions other than formation of a functional PDK1::AKT complex have not been fully elucidated. We fused complementary fragments of intensely fluorescent protein (IFP) to AKT1 and PDK1 to induce a stable complex to study the prerequisites of AKT1 phosphorylation and function. In the stabilized PDK1-IFPC::IFPN-AKT1 complex, AKT1 T308 phosphorylation was independent of PtdIns, as demonstrated by treatment with Phosphatidylinositol 3 Kinase (PI3K) inhibitors. Further when interaction with PtdIns and the cell membrane was prevented by creating PH-domain mutants of AKT1 (R25A) and PDK1 (R474A), AKT1 phosphorylation on T308 was maintained in the PDK1-IFPC::IFPN-AKT1 complex. The PDK1-IFPC::IFPN-AKT1 complex was sufficient for phosphorylation of known AKT substrates, and conferred resistance to inhibitors of PI3K (LY294002, PI103, GDC0941 and TGX286) but not inhibitors of the downstream TORC1 complex (rapamycin). Thus the locus of action of targeted therapeutics can be elucidated by the constitutively active AKT1 complex. Our data indicate that PtdIns and membrane localization are not required for AKT phosphorylation and activation, but rather serve to induce a functional physical interaction between PDK1 and AKT. The PDK1-IFPC::IFPN-AKT1 complex provides a cell-based platform to examine specificity of drugs targeting PI3K pathway components

Raf-independent Deregulation of p38 and JNK Mitogen-activated Protein Kinases Are Critical for Ras Transformation

Activated Ras, but not Raf, causes transformation of RIE-1 epithelial cells, supporting the importance of Raf-independent pathways in mediating Ras transformation. The p38 and JNK mitogen-activated protein kinase cascades are activated by Ras via Raf-independent effector function. Therefore, we determined whether p38 and JNK activation are involved in Ras transformation of RIE-1 epithelial cells. Rather surprisingly, we found that pharmacologic inhibition of p38, together with Raf activation of ERK, was sufficient to mimic the morphologic and growth transformation caused by oncogenic Ras. p38 inhibition together with ERK activation also caused the same alterations in cyclin D1 and p21(CIP1) expression caused by Ras and induced an autocrine growth factor loop important for transformation. Finally, in contrast to p38, we found that JNK activation promoted Ras transformation, and that Ras deregulation of p38 and JNK was not mediated by activation of the Rac small GTPase. We conclude that a key action of Raf-independent effector pathways important for Ras transformation may involve inhibition of p38 and activation of JNK