Background: Kinase Suppressor of Ras (KSR) is a conserved component of the Ras pathway that acts as a molecular scaffold to facilitate signal transmission through the MAPK cascade. Although ...recruitment of KSR1 from the cytosol to the plasma membrane is required for its scaffolding function, the precise mechanism(s) regulating the translocation of KSR1 have not been fully elucidated.
Results: Using mass spectrometry to analyze the KSR1-scaffolding complex, we identify the serine/threonine protein phosphatase PP2A as a KSR1-associated protein and show that PP2A is a critical regulator of KSR1 activity. We find that the enzymatic core subunits of PP2A (PR65A and catalytic C) constitutively associate with the N-terminal domain of KSR1, whereas binding of the regulatory PR55B subunit is induced by growth factor treatment. Specific inhibition of PP2A activity prevents the growth factor-induced dephosphorylation event involved in the membrane recruitment of KSR1 and blocks the activation of KSR1-associated MEK and ERK. Moreover, we find that PP2A activity is required for activation of the Raf-1 kinase and that both Raf and KSR1 must be dephosphorylated by PP2A on critical regulatory 14-3-3 binding sites for KSR1 to promote MAPK pathway activation.
Conclusions: These findings identify KSR1 as novel substrate of PP2A and demonstrate the inducible dephosphorylation of KSR1 in response to Ras pathway activation. Further, these results elucidate a common regulatory mechanism for KSR1 and Raf-1 whereby their localization and activity are modulated by the PP2A-mediated dephosphorylation of critical 14-3-3 binding sites.
Serum starvation stimulates cilia growth in cultured cells, yet serum factors associated with ciliogenesis are unknown. Previously, we showed that starvation induces rapid Rab11-dependent vesicular ...trafficking of Rabin8, a Rab8 guanine-nucleotide exchange factor (GEF), to the mother centriole, leading to Rab8 activation and cilium growth. Here, we demonstrate that through the LPA receptor 1 (LPAR1), serum lysophosphatidic acid (LPA) inhibits Rab11a-Rabin8 interaction and ciliogenesis. LPA/LPAR1 regulates ciliogenesis initiation via downstream PI3K/Akt activation, independent of effects on cell cycle. Akt stabilizes Rab11a binding to its effector, WDR44, and a WDR44-pAkt-phosphomimetic mutant blocks ciliogenesis. WDR44 depletion promotes Rabin8 preciliary trafficking and ciliogenesis-initiating events at the mother centriole. Our work suggests disruption of Akt signaling causes a switch from Rab11-WDR44 to the ciliogenic Rab11-FIP3-Rabin8 complex. Finally, we demonstrate that Akt regulates downstream ciliogenesis processes associated with Rab8-dependent cilia growth. Together, this study uncovers a mechanism whereby serum mitogen signaling regulates Rabin8 preciliary trafficking and ciliogenesis initiation.
•Serum LPA signaling through PI3K/Akt regulates the Rab11-Rab8 ciliogenesis cascade•Akt phosphorylates Rabin8 near its GEF domain, affecting Rab8-dependent cilia assembly•Akt stabilizes Rab11-effector binding to WDR44 by phosphorylating WDR44 at Ser342/344•Effector switch from Rab11-WDR44 to Rab11-FIP3 initiates Rabin8 preciliary trafficking
Cilia assembly is inhibited by activation of the PI3K/Akt pathway by the serum factor lysophosphatidic acid, independent of cell cycle alterations. Walia et al. show that activated Akt phosphorylates WDR44, stabilizing its effector-based binding to Rab11 and preventing Rabin8 preciliary trafficking and ciliogenesis initiation.
Maintenance of genomic integrity is an essential cellular function. We previously reported that the transcription factor and tumor suppressor CCAAT/enhancer binding protein δ (C/EBPδ, CEBPD; also ...known as "NFIL-6β") promotes genomic stability. However, the molecular mechanism was not known. Here, we show that C/EBPδ is a DNA damage-induced gene, which supports survival of mouse bone marrow cells, mouse embryo fibroblasts (MEF), human fibroblasts, and breast tumor cells in response to the DNA cross-linking agent mitomycin C (MMC). Using gene knockout, protein depletion, and overexpression studies, we found that C/EBPδ promotes monoubiquitination of the Fanconi anemia complementation group D2 protein (FANCD2), which is necessary for its function in replication-associated DNA repair. C/EBPδ interacts with FANCD2 and importin 4 (IPO4, also known as "Imp4" and "RanBP4") via separate domains, mediating FANCD2-IPO4 association and augmenting nuclear import of FANCD2, a prerequisite for its monoubiquitination. This study identifies a transcription-independent activity of C/EBPδ in the DNA damage response that may in part underlie its tumor suppressor function. Furthermore, we report a function of IPO4 and nuclear import in the Fanconi anemia pathway of DNA repair.
The metastasis-suppressive activity of Nm23-H1 was previously correlated with its in vitro histidine protein kinase activity, but physiological substrates have not been identified. We hypothesized ...that proteins that
interact with histidine kinases throughout evolution may represent partners for Nm23-H1 and focused on the interaction of
Arabidopsis âtwo-componentâ histidine kinase ERS with CTR1. A mammalian homolog of CTR1 was previously reported to be c-Raf; we now report
that CTR1 also exhibits homology to the kinase suppressor of Ras (KSR), a scaffold protein for the mitogen-activated protein
kinase (MAPK) cascade. Nm23-H1 co-immunoprecipitated KSR from lysates of transiently transfected 293T cells and at endogenous
protein expression levels in MDA-MB-435 breast carcinoma cells. Autophosphorylated recombinant Nm23-H1 phosphorylated KSR
in vitro . Phosphoamino acid analysis identified serine as the major target, and two peaks of Nm23-H1 phosphorylation were identified
upon high performance liquid chromatography analysis of KSR tryptic peptides. Using site-directed mutagenesis, we found that
Nm23-H1 phosphorylated KSR serine 392, a 14-3-3-binding site, as well as serine 434 when serine 392 was mutated. Phosphorylated
MAPK but not total MAPK levels were reduced in an nm23-H1 transfectant of MDA-MB-435 cells. The data identify a complex in vitro histidine-to-serine protein kinase pathway, which may contribute to signal transduction and metastasis.
BRAF is frequently mutated in human cancer and the RASopathy syndromes, with RASopathy mutations often observed in the cysteine-rich domain (CRD). Although the CRD participates in phosphatidylserine ...(PS) binding, the RAS-RAF interaction, and RAF autoinhibition, the impact of these activities on RAF function in normal and disease states is not well characterized. Here, we analyze a panel of CRD mutations and show that they increase BRAF activity by relieving autoinhibition and/or enhancing PS binding, with relief of autoinhibition being the major factor determining mutation severity. Further, we show that CRD-mediated autoinhibition prevents the constitutive plasma membrane localization of BRAF that causes increased RAS-dependent and RAS-independent function. Comparison of the BRAF- and CRAF-CRDs also indicates that the BRAF-CRD is a stronger mediator of autoinhibition and PS binding, and given the increased catalytic activity of BRAF, our studies reveal a more critical role for CRD-mediated autoinhibition in BRAF regulation.
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•RASopathy BRAF-CRD mutations enhance membrane binding and/or relieve autoinhibition•Relief of autoinhibition is the major determinant of BRAF-CRD mutant severity•CRD-mediated autoinhibition is required to prevent aberrant BRAF signaling•BRAF-CRD has greater autoinhibitory and membrane binding activity than the CRAF-CRD
Spencer-Smith et al. demonstrate that RASopathy mutations in the BRAF cysteine-rich domain (CRD) increase BRAF biological activity by relieving autoinhibition and/or enhancing membrane binding. Importantly, they show that CRD-mediated autoinhibition is essential for preventing aberrant BRAF signaling, whereas the CRAF-CRD plays a lesser role in autoinhibition and membrane binding.
RAS signaling: Divide and conquer Holderfield, Matthew; Morrison, Deborah K
Nature chemical biology,
12/2016, Letnik:
13, Številka:
1
Journal Article
Recenzirano
The role of RAS dimerization in tumor biology is an emerging area of preclinical cancer research. Characterization of a RAS monobody indicates that dimer formation is required for RAS signaling, ...revealing the dimer interface as a potential drug target.
The serine/threonine protein kinase encoded by the
Akt proto-oncogene is catalytically inactive in serum-starved primary and immortalized fibroblasts. Here we show that Akt and the Akt-related kinase ...AKT2 are activated by PDGF. The activation was rapid and specific, and it was abrogated by mutations in the Akt Pleckstrin homology (PH) domain. The Akt activation was also shown to depend on PDGFRβ tyrosines Y740 and Y751, which bind phosphatidylinositol 3-kinase (PI 3-kinase) upon phosphorylation. Moreover, Akt activation was blocked by the PI 3-kinase-specific inhibitor wortmanin and the dominant inhibitory N17Ras. Conversely, Akt activity was induced following the addition of phosphatidylinositol-3-phosphate to Akt immunoprecipitates from serum-starved cells in vitro. These results identify Akt as a novel target of PI 3-kinase and suggest that the Akt PH domain may be a mediator of PI 3-kinase signaling.
In the present study, a series of novel madecassic acid derivatives was synthesized and screened against the National Cancer Institute's 60 human cancer cell line panel. Among them, compounds
,
, and
...displayed potent and highly differential antiproliferative activity against 80% of the tumor cells harboring the B-Raf
mutation within the nanomolar range. Structure-activity analysis revealed that a 5-membered A ring containing an α,β-unsaturated aldehyde substituted at C-23 with a 2-furoyl group seems to be crucial to produce this particular growth inhibition signature.
analysis of the cytotoxicity pattern of these compounds identified two highly correlated clinically approved drugs with known B-Raf
inhibitory activity. Follow-up analysis revealed inhibition of the ERK signaling pathway through the reduction of cellular Raf protein levels is a key mechanism of action of these compounds. In particular,
was the most potent compound in suppressing tumor growth of B-Raf
-mutant cell lines and displayed the highest reduction of Raf protein levels among the tested compounds. Taken together, this study revealed that modifications of madecassic acid structure can provide molecules with potent anticancer activity against cell lines harboring the clinically relevant B-Raf
mutation, with compound
identified as a promising lead for the development of new anticancer drugs.
The protein kinase casein kinase 2 (CK2) is a pleiotropic and constitutively active kinase that plays crucial roles in cellular proliferation and survival. Overexpression of CK2, particularly the α ...catalytic subunit (CK2α, CSNK2A1), has been implicated in a wide variety of cancers and is associated with poorer survival and resistance to both conventional and targeted anticancer therapies. Here, we found that CK2α protein is elevated in melanoma cell lines compared with normal human melanocytes. We then tested the involvement of CK2α in drug resistance to Food and Drug Administration-approved single agent targeted therapies for melanoma. In BRAF mutant melanoma cells, ectopic CK2α decreased sensitivity to vemurafenib (BRAF inhibitor), dabrafenib (BRAF inhibitor), and trametinib (MEK inhibitor) by a mechanism distinct from that of mutant NRAS. Conversely, knockdown of CK2α sensitized cells to inhibitor treatment. CK2α-mediated RAF-MEK kinase inhibitor resistance was tightly linked to its maintenance of ERK phosphorylation. We found that CK2α post-translationally regulates the ERK-specific phosphatase dual specificity phosphatase 6 (DUSP6) in a kinase dependent-manner, decreasing its abundance. However, we unexpectedly showed, by using a kinase-inactive mutant of CK2α, that RAF-MEK inhibitor resistance did not rely on CK2α kinase catalytic function, and both wild-type and kinase-inactive CK2α maintained ERK phosphorylation upon inhibition of BRAF or MEK. That both wild-type and kinase-inactive CK2α bound equally well to the RAF-MEK-ERK scaffold kinase suppressor of Ras 1 (KSR1) suggested that CK2α increases KSR facilitation of ERK phosphorylation. Accordingly, CK2α did not cause resistance to direct inhibition of ERK by the ERK1/2-selective inhibitor SCH772984. Our findings support a kinase-independent scaffolding function of CK2α that promotes resistance to RAF- and MEK-targeted therapies.
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