Kinase Suppressor of Ras 2 (KSR2) Regulates Tumor Cell Transformation via AMPK

Fernandez, Mario R.; Henry, Ma Linda D.; Lewis, Robert E.

doi:10.1128/mcb.06754-11

Cited by 48 publications

(59 citation statements)

References 63 publications

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“…MIN6 cells have been used in earlier studies for measurements of cellular bioenergetics [46,47], which indicated that these cells are an appropriate model for these experiments. Our studies indicated that MIN6 β-cells in which ENT3 had been knocked down by transient transfection with siRNAs exhibited significantly higher levels of basal respiration, which was caused both by enhanced ATP-linked respiration and increased proton leak.…”

Section: Discussionmentioning

confidence: 99%

Equilibrative nucleoside transporter 3 depletion in β-cells impairs mitochondrial function and promotes apoptosis: Relationship to pigmented hypertrichotic dermatosis with insulin-dependent diabetes

Liu¹,

Czajka²,

Malik³

et al. 2015

Biochimica et Biophysica Acta (BBA) - Molecular Basis of Diseas

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Loss of function recessive mutations in the SLC29A3 gene that encodes human equilibrative nucleoside transporter 3 (ENT3) have been identified in patients with pigmented hypertrichotic dermatosis with insulin-dependent diabetes (PHID). ENT3 is a member of the equilibrative nucleoside transporter (ENT) family whose primary function is mediating transport of nucleosides and nucleobases. The aims of this study were to characterise ENT3 expression in islet β-cells and identify the effects of its depletion on β-cell mitochondrial activity and apoptosis. RT-PCR amplification identified ENT3 expression in human and mouse islets and exocrine pancreas, and in MIN6 β-cells. Immunohistochemistry using human and mouse pancreas sections exhibited extensive ENT3 immunostaining of β-cells, which was confirmed by co-staining with an anti-insulin antibody. In addition, exposure of dispersed human islet cells and MIN6 β-cells to MitoTracker and an ENT3 antibody showed co-localisation of ENT3 to β-cell mitochondria. Consistent with this, Western blot analysis confirmed enhanced ENT3 immunoreactivity in β-cell mitochondria-enriched fractions. Furthermore, ENT3 depletion in β-cells increased mitochondrial DNA content and promoted an energy crisis characterised by enhanced ATP-linked respiration and proton leak. Finally, inhibition of ENT3 activity by dypridamole and depletion of ENT3 by siRNA-induced knockdown resulted in increased caspase 3/7 activities in β-cells. These observations demonstrate that ENT3 is predominantly expressed by islet β-cells where it co-localises with mitochondria. Depletion of ENT3 causes mitochondrial dysfunction which is associated with enhanced β-cell apoptosis. Thus, apoptotic loss of islet β-cells may contribute to the occurrence of autoantibody-negative insulin-dependent diabetes in individuals with non-functional ENT3 mutations.

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Section: Discussionmentioning

confidence: 99%

Equilibrative nucleoside transporter 3 depletion in β-cells impairs mitochondrial function and promotes apoptosis: Relationship to pigmented hypertrichotic dermatosis with insulin-dependent diabetes

Liu¹,

Czajka²,

Malik³

et al. 2015

Biochimica et Biophysica Acta (BBA) - Molecular Basis of Diseas

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“…Ras V12 -activated AMPK␣1/␣2 null MEFs are resistant to tumor formation in vivo, with increased susceptibility to anoikis (36,37), and KSR2-dependent MEF transformation requires AMPK activation (38). AMPK activity is increased in early stages of gliomas (39).…”

Section: Discussionmentioning

confidence: 99%

AMPK Promotes Aberrant PGC1β Expression To Support Human Colon Tumor Cell Survival

Fisher

Das

Kim

et al. 2015

Molecular and Cellular Biology

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A major goal of cancer research is the identification of tumor-specific vulnerabilities that can be exploited for the development of therapies that are selectively toxic to the tumor. We show here that the transcriptional coactivators peroxisome proliferatoractivated receptor gamma coactivator 1␤ (PGC1␤) and estrogen-related receptor ␣ (ERR␣) are aberrantly expressed in human colon cell lines and tumors. With kinase suppressor of Ras 1 (KSR1) depletion as a reference standard, we used functional signature ontology (FUSION) analysis to identify the ␥1 subunit of AMP-activated protein kinase (AMPK) as an essential contributor to PGC1␤ expression and colon tumor cell survival. Subsequent analysis revealed that a subunit composition of AMPK (␣2␤2␥1) is preferred for colorectal cancer cell survival, at least in part, by stabilizing the tumor-specific expression of PGC1␤. In contrast, PGC1␤ and ERR␣ are not detectable in nontransformed human colon epithelial cells, and depletion of the AMPK␥1 subunit has no effect on their viability. These data indicate that Ras oncogenesis relies on the aberrant activation of a PGC1␤-dependent transcriptional pathway via a specific AMPK isoform.A third of all human cancers, including a substantial percentage of colorectal, lung, and pancreatic cancers, are driven by activating mutations in Ras genes. Activating K-Ras mutations are present in 35 to 40% of colon tumors and are thought to be both drivers of tumorigenesis and determinants of therapeutic regimens (1). Therapeutic disruption of Ras function has been clinically ineffective to date, but investigation of Ras pleiotropy continues to yield a diversity of downstream effectors with obligate roles in the maintenance and adaptation of Ras-driven tumors to changing environments. The Raf-MEK-extracellular signal-regulated kinase (ERK) signaling pathway is essential for the oncogenic properties of mutated K-Ras (2). However, numerous potent and specific MEK inhibitors have been developed yet have failed to demonstrate single-agent efficacy in cancer treatment (3). As a molecular scaffold of the Raf-MEK-ERK kinase cascade (4, 5), kinase suppressor of Ras 1 (KSR1) is necessary and sufficient for Ras V12 -induced tumorigenesis (4), mouse embryo fibroblast (MEF) transformation (5, 6), and pancreatic cancer growth (7) but dispensable for normal development (4). KSR1 is overexpressed in endometrial carcinoma and is required for both proliferation and anchorage-independent growth of endometrial cancer cells (8). Except for minor defects in hair follicles, KSR1 knockout mice are fertile and develop normally (4).This observation predicts that small molecules targeting KSR1 and functionally related effectors should preferentially target Rasdriven tumors while leaving normal tissue largely unaffected. More generally, this observation demonstrates that tumor cells, while under selective pressure to adapt to inhospitable environments and proliferate without constraint, will adopt strategies that, while advantageous to that singular purpose, create...

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“…The shRNA for Rap1a, Rap1b, B-Raf, and C-Raf were described previously (13,16,(63)(64)(65)(66). The sense KSR1 shRNA oligonucleotide is GTGC-CAGAAGAGCATGATA (human sequence, corresponding to the mouse KSR1 siRNA) (67).…”

Section: Methodsmentioning

confidence: 99%

Phosphorylation of Rap1 by cAMP-dependent Protein Kinase (PKA) Creates a Binding Site for KSR to Sustain ERK Activation by cAMP

Takahashi

Dillon

et al. 2017

Journal of Biological Chemistry

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Edited by Jeffrey E. PessinCyclic adenosine monophosphate (cAMP) is an important mediator of hormonal stimulation of cell growth and differentiation through its activation of the extracellular signal-regulated kinase (ERK) cascade. Two small G proteins, Ras and Rap1 have been proposed to mediate this activation. Using HEK293 cells as a model system, we have recently shown that both Ras and Rap1 are required for cAMP signaling to ERKs. However, cAMP-dependent Ras signaling to ERKs is transient and rapidly terminated by PKA phosphorylation of the Raf isoforms C-Raf and B-Raf. In contrast, cAMP-dependent Rap1 signaling to ERKs and Rap1 is potentiated by PKA. We show that this is due to sustained binding of B-Raf to Rap1. One of the targets of PKA is Rap1 itself, directly phosphorylating Rap1a on serine 180 and Rap1b on serine 179. We show that these phosphorylations create potential binding sites for the adaptor protein 14-3-3 that links Rap1 to the scaffold protein KSR. These results suggest that Rap1 activation of ERKs requires PKA phosphorylation and KSR binding. Because KSR and B-Raf exist as heterodimers within the cell, this binding also brings B-Raf to Rap1, allowing Rap1 to couple to ERKs through B-Raf binding to Rap1 independently of its Ras-binding domain.Hormones that are coupled to the second messenger cyclic adenosine monophosphate (cAMP) signal to multiple intracellular pathways. One of these, the MAP kinase, or ERK (extracellular signal-regulated kinase) cascade, mediates many physiological functions of cAMP in development (1), metabolism (2, 3), cognition (4 -6), and cell growth (7-12). In all cases, cAMP signaling to ERKs requires the recruitment of the MAP kinase kinase kinase Raf to one of two small G proteins, Ras or Rap1. The ability to signal to both Ras and Rap1 allows cAMP to trigger the sustained activation of ERKs, which is required for many physiological functions, including the transcription/stabilization of many transcription factors (13,14).In addition to its ability to activate both Ras and Rap1, cAMP can inhibit signaling to Ras. PKA antagonizes Ras signaling through its phosphorylation of the Raf isoform C-Raf on serine 259 (Ser-259) (15). This phosphorylation and subsequent recruitment of 14-3-3 result in the release of C-Raf from active Ras (15, 16). We have recently shown that PKA phosphorylation of the homologous site in B-Raf (Ser-365) similarly prevents the direct binding of B-Raf to Ras (16). Despite this phosphorylation of B-Raf, B-Raf is still capable of binding to Rap1. This is physiologically relevant, as Rap1/B-Raf links PKA to ERKs (17, 18). How Rap1 is able to couple to B-Raf following PKA phosphorylation of B-Raf is unknown.We and others have identified Rap1 as a target for phosphorylation by PKA (19 -21). Here, we show that Rap1 phosphorylation plays a role in the ability of B-Raf to remain bound to Rap1 following cAMP stimulation. Rap1 phosphorylation creates a novel 14-3-3 binding site that connects Rap1 to the scaffold KSR. Because KSR can exist as a dimer with B-...

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Kinase Suppressor of Ras 2 (KSR2) Regulates Tumor Cell Transformation via AMPK

Cited by 48 publications

References 63 publications

Equilibrative nucleoside transporter 3 depletion in β-cells impairs mitochondrial function and promotes apoptosis: Relationship to pigmented hypertrichotic dermatosis with insulin-dependent diabetes

Equilibrative nucleoside transporter 3 depletion in β-cells impairs mitochondrial function and promotes apoptosis: Relationship to pigmented hypertrichotic dermatosis with insulin-dependent diabetes

AMPK Promotes Aberrant PGC1β Expression To Support Human Colon Tumor Cell Survival

Phosphorylation of Rap1 by cAMP-dependent Protein Kinase (PKA) Creates a Binding Site for KSR to Sustain ERK Activation by cAMP

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