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Bcl-2 does not require Raf kinase activity for its death-protective function.

ABSTRACT: It has been widely accepted that the oncogene product bcl-2 protects mammalian cells from programmed cell death (apoptosis). The molecules and signalling pathways upon which bcl-2 acts are, however, still ill-defined. Recently, bcl-2 was shown to interact with c-raf-1 in vitro. Furthermore, an active form of c-raf-1 delayed apoptosis induced by trophic factor deprivation and enhanced the death-suppressive function of bcl-2 when co-expressed. This has led to the hypothesis that bcl-2 communicates cell-death protection via a raf-dependent signal transduction pathway. Here we show, by various immunological and biochemical methods, that bcl-2 does not stably associate with c-raf-1 in cellular extracts prepared from fibroblasts before or after treatment with agents that induce apoptosis. Unexpectedly, bcl-2 function is entirely maintained, if not improved, when raf-dependent signalling is experimentally abrogated. In fact, bcl-2 allows the stable overexpression of a kinase-defective dominant-negative raf mutant that usually interferes with cell viability and/or proliferation. Our results indicate that bcl-2 does not require c-raf-1 kinase activity and an associated mitogen-activated protein kinase signalling pathway for its survival function. This property may be exploited to dissect cellular events that are dependent or independent of c-raf-1 kinase activity.

SUBMITTER: Olivier R

PROVIDER: S-EPMC1218403 | biostudies-other | 1997 May

REPOSITORIES: biostudies-other

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Diacylglycerol kinase eta augments C-Raf activity and B-Raf/C-Raf heterodimerization.

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Nuclear speckle integrity and function require TAO2 kinase.

Project description:Nuclear speckles are non-membrane-bound organelles known as storage sites for messenger RNA (mRNA) processing and splicing factors. More recently, nuclear speckles have also been implicated in splicing and export of a subset of mRNAs, including the influenza virus M mRNA that encodes proteins required for viral entry, trafficking, and budding. However, little is known about how nuclear speckles are assembled or regulated. Here, we uncovered a role for the cellular protein kinase TAO2 as a constituent of nuclear speckles and as a factor required for the integrity of these nuclear bodies and for their functions in pre-mRNA splicing and trafficking. We found that a nuclear pool of TAO2 is localized at nuclear speckles and interacts with nuclear speckle factors involved in RNA splicing and nuclear export, including SRSF1 and Aly/Ref. Depletion of TAO2 or inhibition of its kinase activity disrupts nuclear speckle structure, decreasing the levels of several proteins involved in nuclear speckle assembly and splicing, including SC35 and SON. Consequently, splicing and nuclear export of influenza virus M mRNA were severely compromised and caused a disruption in the virus life cycle. In fact, low levels of TAO2 led to a decrease in viral protein levels and inhibited viral replication. Additionally, depletion or inhibition of TAO2 resulted in abnormal expression of a subset of mRNAs with key roles in viral replication and immunity. Together, these findings uncovered a function of TAO2 in nuclear speckle formation and function and revealed host requirements and vulnerabilities for influenza infection.

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Death Receptor 5 Networks Require Membrane Cholesterol for Proper Structure and Function.

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Single substitution within the RKTR motif impairs kinase activity but promotes dimerization of RAF kinase.

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Project description:By virtue of their ability to induce apoptosis and regulate growth, differentiation, and cytokine responses, the tumor necrosis factor receptor (TNFR) superfamily members have emerged as attractive targets for anticancer therapeutics. Agonistic antibodies to apoptosis-inducing TNFRs, such as death receptor 5 (DR5), although displaying impressive activities against a variety of tumors in preclinical models, appear to be less active in clinical trials. We report that the in vivo apoptotic and antitumor activities of these antibodies have an absolute requirement for the coengagement of an inhibitory Fcγ receptor, FcγRIIB. Anti-DR5 antibodies of the type currently in clinical trials have weak FcγRIIB binding and thus are compromised in their proapoptotic and antitumor activities in both colon and breast carcinoma models. Enhancing FcγRIIB engagement increases apoptotic and antitumor potency. Our results demonstrate that Fc domain interactions are critical to the therapeutic activity of anti-DR5 antibodies and, together with previous reports on agonistic anti-CD40 antibodies, establish a common requirement for FcγRIIB coengagement for optimal biological effects of agonistic anti-TNFR antibodies.

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Endosomal targeting of MEK2 requires RAF, MEK kinase activity and clathrin-dependent endocytosis.

Project description:To study spatiotemporal regulation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK1/2) signaling cascade in living cells, a HeLa cell line in which MAPK kinase of ERK kinase (MEK) 2 (MAPK kinase) was knocked down by RNA interference and replaced with the green fluorescent protein (GFP)-tagged MEK2 was generated. In these cells, MEK2-GFP was stably expressed at a level similar to that of the endogenous MEK2 in the parental cells. Upon activation of the EGF receptor (EGFR), a pool of MEK2-GFP was found initially translocated to the plasma membrane and then accumulated in a subset of early and late endosomes. However, activated MEK was detected only at the plasma membrane and not in endosomes. Surprisingly, MEK2-GFP endosomes did not contain active EGFR, suggesting that endosomal MEK2-GFP was separated from the upstream signaling complexes. Knockdown of clathrin by small interfering RNA (siRNA) abolished MEK2 recruitment to endosomes but resulted in increased activation of ERK without affecting the activity of MEK2-GFP. The accumulation of MEK2-GFP in endosomes was also blocked by siRNA depletion of RAF kinases and by the MEK1/2 inhibitor, UO126. We propose that the recruitment of MEK2 to endosomes can be a part of the negative feedback regulation of the EGFR-MAPK signaling pathway by endocytosis.

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Discovery of a selective inhibitor of oncogenic B-Raf kinase with potent antimelanoma activity.

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