Krishnaraj Rajalingam

Prohibitin is required for Ras-induced Raf–MEK–ERK activation and epithelial cell migration

Ras proteins control the signalling pathways that are responsible for normal growth and malignant transformation. Raf protein kinases are direct Ras effector proteins that initiate the mitogen-activated protein kinase (MAPK) cascade, which mediates diverse biological functions such as cell growth, survival and differentiation. Here we show that prohibitin, a ubiquitously expressed and evolutionarily conserved protein is indispensable for the activation of the Raf–MEK–ERK pathway by Ras. The membrane targeting and activation of C-Raf by Ras needs prohibitin in vivo. In addition, direct interaction with prohibitin is required for C-Raf activation. C-Raf kinase fails to interact with the active Ras induced by epidermal growth factor in the absence of prohibitin. Moreover, in prohibitin-deficient cells the adhesion complex proteins cadherin and β-catenin relocalize to the plasma membrane and thereby stabilize adherens junctions. Our data show an unexpected role of prohibitin in the activation of the Ras–Raf signalling pathway and in modulating epithelial cell adhesion and migration.

Oncogenic ERBB3 Mutations in Human Cancers

The human epidermal growth factor receptor (HER) family of tyrosine kinases is deregulated in multiple cancers either through amplification, overexpression, or mutation. ERBB3/HER3, the only member with an impaired kinase domain, although amplified or overexpressed in some cancers, has not been reported to carry oncogenic mutations. Here, we report the identification of ERBB3 somatic mutations in ~11% of colon and gastric cancers. We found that the ERBB3 mutants transformed colonic and breast epithelial cells in a ligand-independent manner. However, the mutant ERBB3 oncogenic activity was dependent on kinase-active ERBB2. Furthermore, we found that anti-ERBB antibodies and small molecule inhibitors effectively blocked mutant ERBB3-mediated oncogenic signaling and disease progression in vivo.

Epithelial Cells Infected with Chlamydophila pneumoniae (Chlamydia pneumoniae) Are Resistant to Apoptosis

ABSTRACT The obligate intracellular pathogen Chlamydophila pneumoniae (Chlamydia pneumoniae) initiates infections in humans via the mucosal epithelia of the respiratory tract. Here, we report that epithelial cells infected with C. pneumoniae are resistant to apoptosis induced by treatment with drugs or by death receptor ligation. The induction of protection from apoptosis depended on the infection conditions since only cells containing large inclusions were protected. The underlying mechanism of infection-induced apoptosis resistance probably involves mitochondria, the major integrators of apoptotic signaling. In the infected cells, mitochondria did not respond to apoptotic stimuli by the release of apoptogenic factors required for the activation of caspases. Consequently, active caspase-3 was absent in infected cells. Our data suggest a direct modulation of apoptotic pathways in epithelial cells by C. pneumoniae.

IAPs regulate the plasticity of cell migration by directly targeting Rac1 for degradation

Inhibitors of apoptosis proteins (IAPs) are a highly conserved class of multifunctional proteins. Rac1 is a well‐studied Rho GTPase that controls numerous basic cellular processes. While the regulation of nucleotide binding to Rac1 is well understood, the molecular mechanisms controlling Rac1 degradation are not known. Here, we demonstrate X‐linked IAP (XIAP) and cellular IAP1 (c‐IAP1) directly bind to Rac1 in a nucleotide‐independent manner to promote its polyubiquitination at Lys147 and proteasomal degradation. These IAPs are also required for degradation of Rac1 upon CNF1 toxin treatment or RhoGDI depletion. Consistently, downregulation of XIAP or c‐IAP1 by various strategies led to an increase in Rac1 protein levels in primary and tumour cells, leading to an elongated morphology and enhanced cell migration. Further, XIAP counteracts Rac1‐dependent cellular polarization in the developing zebrafish hindbrain and promotes the delamination of neurons from the normal tissue architecture. These observations unveil an evolutionarily conserved role of IAPs in controlling Rac1 stability thereby regulating the plasticity of cell migration and morphogenesis.

Ras-Raf Signaling Needs Prohibitin

Prohibitin has been connected to diverse cellular functions including cell cycle control, senescence, apoptosis and stabilization of mitochondrial proteins. By employing a loss of function approach using siRNAs we have demonstrated an unexpected role of PHB in the activation of Raf/MEK/ERK pathway by active Ras and in modulating epithelial cell adhesion and migration. PHB directly interacts with C-Raf and is required for the displacement of 14-3-3 from C-Raf by active Ras to facilitate plasma membrane localization and activation. Further, the adherent complex proteins cadherin and β-catenin were localized to plasma membrane suggesting stabilized adherent junctions in PHB silenced cells. Our findings demonstrated a function of PHB in the control of a central signaling pathway involved in cell growth and malignant transformation.

X-linked and cellular IAPs modulate the stability of C-RAF kinase and cell motility

Inhibitor of apoptosis proteins (IAP) are evolutionarily conserved anti-apoptotic regulators. C-RAF protein kinase is a direct RAS effector protein, which initiates the classical mitogen-activated protein kinase (MAPK) cascade. This signalling cascade mediates diverse biological functions, such as cell growth, proliferation, migration, differentiation and survival. Here we demonstrate that XIAP and c-IAPs bind directly to C-RAF kinase and that siRNA-mediated silencing of XIAP and c-IAPs leads to stabilization of C-RAF in human cells. XIAP binds strongly to C-RAF and promotes the ubiquitylation of C-RAF in vivo through the Hsp90-mediated quality control system, independently of its E3 ligase activity. In addition, XIAP or c-IAP-1/2 knockdown cells showed enhanced cell migration in a C-RAF-dependent manner. XIAP promotes binding of CHIP (carboxy terminal Hsc70-interacting protein), a chaperone-associated ubiquitin ligase, to the C-RAF–Hsp90 complex in vivo. Interfering with CHIP expression resulted in stabilization of C-RAF and enhanced cell migration, as observed in XIAP knockdown cells. Our data show an unexpected role of XIAP and c-IAPs in the turnover of C-RAF protein, thereby modulating the MAPK signalling pathway and cell migration.

IAP-IAP Complexes Required for Apoptosis Resistance of C. trachomatis-Infected Cells

Host cells infected with obligate intracellular bacteria Chlamydia trachomatis are profoundly resistant to diverse apoptotic stimuli. The molecular mechanisms underlying the block in apoptotic signaling of infected cells is not well understood. Here we investigated the molecular mechanism by which apoptosis induced via the tumor necrosis factor (TNF) receptor is prevented in infected epithelial cells. Infection with C. trachomatis leads to the up-regulation of cellular inhibitor of apoptosis (cIAP)-2, and interfering with cIAP-2 up-regulation sensitized infected cells for TNF-induced apoptosis. Interestingly, besides cIAP-2, cIAP-1 and X-linked IAP, although not differentially regulated by infection, are required to maintain apoptosis resistance in infected cells. We detected that IAPs are constitutively organized in heteromeric complexes and small interfering RNA–mediated silencing of one of these IAPs affects the stability of another IAP. In particular, the stability of cIAP-2 is modulated by the presence of X-linked IAP and their interaction is stabilized in infected cells. Our observations suggest that IAPs are functional and stable as heteromers, a thus far undiscovered mechanism of IAP regulation and its role in modulation of apoptosis.

Mcl-1 Is a Key Regulator of Apoptosis Resistance in Chlamydia trachomatis-Infected Cells

Chlamydia are obligate intracellular bacteria that cause variety of human diseases. Host cells infected with Chlamydia are protected against many different apoptotic stimuli. The induction of apoptosis resistance is thought to be an important immune escape mechanism allowing Chlamydia to replicate inside the host cell. Infection with C. trachomatis activates the Raf/MEK/ERK pathway and the PI3K/AKT pathway. Here we show that inhibition of these two pathways by chemical inhibitors sensitized C. trachomatis infected cells to granzyme B-mediated cell death. Infection leads to the Raf/MEK/ERK-mediated up-regulation and PI3K-dependent stabilization of the anti-apoptotic Bcl-2 family member Mcl-1. Consistently, interfering with Mcl-1 up-regulation sensitized infected cells for apoptosis induced via the TNF receptor, DNA damage, granzyme B and stress. Our data suggest that Mcl-1 up-regulation is primarily required to maintain apoptosis resistance in C. trachomatis-infected cells.

Flotillin-1/reggie-2 protein plays dual role in activation of receptor-tyrosine kinase/mitogen-activated protein kinase signaling.

Background: We have analyzed the role of flotillins during EGF receptor signaling. Results: Flotillin-1 knockdown results in impaired activation of EGFR and MAP kinases, with which flotillin-1 interacts. Conclusion: Flotillin-1 may be a novel scaffolding protein in MAP kinase signaling. Significance: Flotillin-1 is essential for proper MAP kinase signaling. Our previous work has shown that the membrane microdomain-associated flotillin proteins are potentially involved in epidermal growth factor (EGF) receptor signaling. Here we show that knockdown of flotillin-1/reggie-2 results in reduced EGF-induced phosphorylation of specific tyrosines in the EGF receptor (EGFR) and in inefficient activation of the downstream mitogen-activated protein (MAP) kinase and Akt signaling. Although flotillin-1 has been implicated in endocytosis, its depletion affects neither the endocytosis nor the ubiquitination of the EGFR. However, EGF-induced clustering of EGFR at the cell surface is altered in cells lacking flotillin-1. Furthermore, we show that flotillins form molecular complexes with EGFR in an EGF/EGFR kinase-independent manner. However, knockdown of flotillin-1 appears to affect the activation of the downstream MAP kinase signaling more directly. We here show that flotillin-1 forms a complex with CRAF, MEK1, ERK, and KSR1 (kinase suppressor of RAS) and that flotillin-1 knockdown leads to a direct inactivation of ERK1/2. Thus, flotillin-1 plays a direct role during both the early phase (activation of the receptor) and late (activation of MAP kinases) phase of growth factor signaling. Our results here unveil a novel role for flotillin-1 as a scaffolding factor in the regulation of classical MAP kinase signaling. Furthermore, our results imply that other receptor-tyrosine kinases may also rely on flotillin-1 upon activation, thus suggesting a general role for flotillin-1 as a novel factor in receptor-tyrosine kinase/MAP kinase signaling.

Regulation of RAF activity by 14-3-3 proteins: RAF kinases associate functionally with both homo- and heterodimeric forms of 14-3-3 proteins.

Mammalian 14-3-3 proteins play a crucial role in the activation process of RAF kinases. However, little is known about the selectivity of the mammalian 14-3-3 isoforms with respect to RAF association and activation. Using mass spectrometry, we analyzed the composition of the 14-3-3 isoforms attached to RAF kinases and found that B-RAF associates in vivo with 14-3-3 at much higher diversity than A- and C-RAF. We also examined in vitro binding of purified mammalian 14-3-3 proteins to RAF kinases using surface plasmon resonance techniques. While B- and C-RAF exhibited binding to all seven 14-3-3 isoforms, A-RAF bound with considerably lower affinities to ϵ, τ, and σ 14-3-3. These findings indicate that 14-3-3 proteins associate with RAF isoforms in a pronounced isoform-specific manner. Because 14-3-3 proteins appear in dimeric forms, we addressed the question of whether both homo- and heterodimeric forms of 14-3-3 proteins participate in RAF signaling. For that purpose, the budding yeast Saccharomyces cerevisiae, possessing only two 14-3-3 isoforms (BMH1 and BMH2), served as testing system. By deletion of the single BMH2 gene, we found that both homo- and heterodimeric forms of 14-3-3 can participate in RAF activation. Furthermore, we show that A-, B-, and C-RAF activity is differentially regulated by its C-terminal and internal 14-3-3 binding domain. Finally, prohibitin, a scaffold protein that affects C-RAF activation in a stimulatory manner, proved to interfere with the internal 14-3-3 binding site in C-RAF. Together, our results shed more light on the complex mechanism of RAF activation, particularly with respect to activation steps that are mediated by 14-3-3 proteins and prohibitin.