Human Epidermal Growth Factor Receptor 2 (HER2) Impedes MLK3 Kinase Activity to Support Breast Cancer Cell Survival.

Human epidermal growth factor receptor 2 (HER2) is amplified in ∼ 15-20% of human breast cancer and is important for tumor etiology and therapeutic options of breast cancer. Up-regulation of HER2 oncogene initiates cascades of events cumulating to the stimulation of transforming PI3K/AKT signaling, which also plays a dominant role in supporting cell survival and efficacy of HER2-directed therapies. Although investigating the underlying mechanisms by which HER2 promotes cell survival, we noticed a profound reduction in the kinase activity of a pro-apoptotic mixed lineage kinase 3 (MLK3) in HER2-positive (HER2+) but not in HER2-negative (HER2-) breast cancer tissues, whereas both HER2+ and HER2- tumors expressed a comparable level of MLK3 protein.

TGIF governs a feed-forward network that empowers Wnt signaling to drive mammary tumorigenesis.

Many types of human cancers having hyperactivated Wnt signaling display no causative alterations in known effectors of this pathway. Here, we report a function of TGIF in Wnt signaling. TGIF associates with and diverts Axin1 and Axin2 from the β-catenin destruction complex, therefore allowing β-catenin accrual.

Overexpression of 14-3-3ζ Increases Brain Levels of C/EBP Homologous Protein CHOP.

Recent studies demonstrated that overexpression of the molecular chaperone 14-3-3ζ protects the brain against endoplasmic reticulum (ER) stress and prolonged seizures. The 14-3-3 targets responsible for improved neuronal survival after seizures remain unknown. Here we explored the mechanism, finding that protein levels of the ER-stress-associated transcription factor C/EBP homologous protein (CHOP) were significantly higher in 14-3-3ζ-overexpressing mice.

AKT and 14-3-3 regulate Notch4 nuclear localization.

Members of the Notch family of transmembrane receptors, Notch1-4 in mammals, are involved in the regulation of cell fate decisions and cell proliferation in various organisms. The Notch4 isoform, which is specific to mammals, was originally identified as a viral oncogene in mice, Int3, able to initiate mammary tumors. In humans, Notch4 expression appears to be associated with breast cancer stem cells and endocrine resistance.

Disruption of the PHRF1 Tumor Suppressor Network by PML-RARα Drives Acute Promyelocytic Leukemia Pathogenesis.

PHRF1 functions as an essential component of the TGF-β tumor suppressor pathway by triggering degradation of the homeodomain repressor factor TGIF. This leads to redistribution of cPML into the cytoplasm, where it coordinates phosphorylation and activation of Smad2 by the TGF-β receptor. In acute promyelocytic leukemia (APL), acquisition of PML-RARα is known to impede critical aspects of TGF-β signaling, including myeloid differentiation.

Resveratrol regulates PTEN/Akt pathway through inhibition of MTA1/HDAC unit of the NuRD complex in prostate cancer.

Metastasis associated protein 1 (MTA1) is a component of the nucleosome remodeling and deacetylating (NuRD) complex which mediates gene silencing and is overexpressed in several cancers. We reported earlier that resveratrol, a dietary stilbene found in grapes, can down-regulate MTA1. In the present study, we show that PTEN is inactivated by MTA1 in prostate cancer cells.

Sirt2 deacetylase is a novel AKT binding partner critical for AKT activation by insulin.

AKT/PKB kinases transmit insulin and growth factor signals downstream of phosphatidylinositol 3-kinase (PI3K). AKT activation involves phosphorylation at two residues, Thr(308) and Ser(473), mediated by PDK1 and the mammalian target of rapamycin complex 2 (mTORC2), respectively. Impaired AKT activation is a key factor in metabolic disorders involving insulin resistance, whereas hyperactivation of AKT is linked to cancer pathogenesis.

MEK-1 activates C-Raf through a Ras-independent mechanism.

C-Raf is a member of the Ras-Raf-MEK-ERK mitogen-activated protein kinase (MAPK) signaling pathway that plays key roles in diverse physiological processes and is upregulated in many human cancers. C-Raf activation involves binding to Ras, increased phosphorylation and interactions with co-factors. Here, we describe a Ras-independent in vivo pathway for C-Raf activation by its downstream target MEK.

Transgenic overexpression of 14-3-3 zeta protects hippocampus against endoplasmic reticulum stress and status epilepticus in vivo.

14-3-3 proteins are ubiquitous molecular chaperones that are abundantly expressed in the brain where they regulate cell functions including metabolism, the cell cycle and apoptosis. Brain levels of several 14-3-3 isoforms are altered in diseases of the nervous system, including epilepsy. The 14-3-3 zeta (ζ) isoform has been linked to endoplasmic reticulum (ER) function in neurons, with reduced levels provoking ER stress and increasing vulnerability to excitotoxic injury.

Mixed-lineage kinase 3 phosphorylates prolyl-isomerase Pin1 to regulate its nuclear translocation and cellular function.

Nuclear protein peptidyl-prolyl isomerase Pin1-mediated prolyl isomerization is an essential and novel regulatory mechanism for protein phosphorylation. Therefore, tight regulation of Pin1 localization and catalytic activity is crucial for its normal nuclear functions. Pin1 is commonly dysregulated during oncogenesis and likely contributes to these pathologies; however, the mechanism(s) by which Pin1 catalytic activity and nuclear localization are increased is unknown.

FoxO transcription factors; Regulation by AKT and 14-3-3 proteins.

The forkhead box O (FoxO) transcription factor family is a key player in an evolutionary conserved pathway downstream of insulin and insulin-like growth factor receptors. The mammalian FoxO family consists of FoxO1, 3, 4 and 6, which share high similarity in their structure, function and regulation. FoxO proteins are involved in diverse cellular and physiological processes including cell proliferation, apoptosis, reactive oxygen species (ROS) response, longevity, cancer and regulation of cell cycle and metabolism.

Bimodal regulation of FoxO3 by AKT and 14-3-3.

FoxO3 is a member of FoxO family transcription factors that mediate cellular functions downstream of AKT. FoxO3 phosphorylation by AKT generates binding sites for 14-3-3, which in-turn regulates FoxO3 transcriptional activity and localization. We examine here the functional significance of AKT-FoxO3 interaction and further detail the mechanistic aspects of FoxO3 regulation by AKT and 14-3-3.

Posttranslational regulation of membrane type 1-matrix metalloproteinase (MT1-MMP) in mouse PTEN null prostate cancer cells: Enhanced surface expression and differential O-glycosylation of MT1-MMP.

Membrane type 1 (MT1)-matrix metalloproteinase (MT1-MMP) is a membrane-tethered MMP that has been shown to play a key role in promoting cancer cell invasion. MT1-MMP is highly expressed in bone metastasis of prostate cancer (PC) patients and promotes intraosseous tumor growth of PC cells in mice. The majority of metastatic prostate cancers harbor loss-of-function mutations or deletions of the tumor suppressor PTEN (phosphatase and tensin homologue deleted on chromosome ten).

Life span extension and neuronal cell protection by Drosophila nicotinamidase.

The life span of model organisms can be modulated by environmental conditions that influence cellular metabolism, oxidation, or DNA integrity. The yeast nicotinamidase gene pnc1 was identified as a key transcriptional target and mediator of calorie restriction and stress-induced life span extension. PNC1 is thought to exert its effect on yeast life span by modulating cellular nicotinamide and NAD levels, resulting in increased activity of Sir2 family class III histone deacetylases.

Raf kinases: function, regulation and role in human cancer.

The Ras-Raf-MAPK pathway regulates diverse physiological processes by transmitting signals from membrane based receptors to various nuclear, cytoplasmic and membrane-bound targets, coordinating a large variety of cellular responses. Function of Raf family kinases has been shown to play a role during organism development, cell cycle regulation, cell proliferation and differentiation, cell survival and apoptosis and many other cellular and physiological processes. Aberrations along the Ras-Raf-MAPK pathway play an integral role in various biological processes concerning human health and disease.

Distinct activities of the related protein kinases Cdk1 and Ime2.

In budding yeast, commitment to DNA replication during the normal cell cycle requires degradation of the cyclin-dependent kinase (CDK) inhibitor Sic1. The G1 cyclin-CDK complexes Cln1-Cdk1 and Cln2-Cdk1 initiate the process of Sic1 removal by directly catalyzing Sic1 phosphorylation at multiple sites. Commitment to DNA replication during meiosis also appears to require Sic1 degradation, but the G1 cyclin-CDK complexes are not involved.

Microphthalmia-associated transcription factor interactions with 14-3-3 modulate differentiation of committed myeloid precursors.

The microphthalmia-associated transcription factor (MITF) is required for terminal osteoclast differentiation and is a target for signaling pathways engaged by colony stimulating factor (CSF)-1 and receptor-activator of nuclear factor-kappaB ligand (RANKL). Work presented here demonstrates that MITF can shuttle from cytoplasm to nucleus dependent upon RANKL/CSF-1 action. 14-3-3 was identified as a binding partner of MITF in osteoclast precursors, and overexpression of 14-3-3 in a transgenic model resulted in increased cytosolic localization of MITF and decreased expression of MITF target genes.

14-3-3 proteins as potential oncogenes.

14-3-3 proteins are a family of highly conserved cellular proteins that play key roles in the regulation of central physiological pathways. More than 200 14-3-3 target proteins have been identified, including proteins involved in mitogenic and cell survival signaling, cell cycle control and apoptotic cell death. Importantly, the involvement of 14-3-3 proteins in the regulation of various oncogenes and tumor suppressor genes points to a potential role in human cancer.

Identification of novel in vivo Raf-1 phosphorylation sites mediating positive feedback Raf-1 regulation by extracellular signal-regulated kinase.

The Ras-Raf-mitogen-activated protein kinase cascade is a key growth-signaling pathway, which uncontrolled activation results in transformation. Although the exact mechanisms underlying Raf-1 regulation remain incompletely understood, phosphorylation has been proposed to play a critical role in this regulation. We report here three novel epidermal growth factor-induced in vivo Raf-1 phosphorylation sites that mediate positive feedback Raf-1 regulation.

Identification of Raf-1 S471 as a novel phosphorylation site critical for Raf-1 and B-Raf kinase activities and for MEK binding.

The Ras-Raf-MAPK cascade is a key growth-signaling pathway and its uncontrolled activation results in cell transformation. Although the general features of the signal transmission along the cascade are reasonably defined, the mechanisms underlying Raf activation remain incompletely understood. Here, we show that Raf-1 dephosphorylation, primarily at epidermal growth factor (EGF)-induced sites, abolishes Raf-1 kinase activity.

Significance of 14-3-3 self-dimerization for phosphorylation-dependent target binding.

14-3-3 proteins via binding serine/threonine-phosphorylated proteins regulate diverse intracellular processes in all eukaryotic organisms. Here, we examine the role of 14-3-3 self-dimerization in target binding, and in the susceptibility of 14-3-3 to undergo phosphorylation. Using a phospho-specific antibody developed against a degenerated mode-1 14-3-3 binding motif (RSxpSxP), we demonstrate that most of the 14-3-3-associated proteins in COS-7 cells are phosphorylated on sites that react with this antibody.

Cross-talk between JNK/SAPK and ERK/MAPK pathways: sustained activation of JNK blocks ERK activation by mitogenic factors.

Mixed lineage kinases (MLKs) are a family of serine/threonine kinases that function in the SAPK signaling cascade. MLKs activate JNK/SAPK in vivo by directly phosphorylating and activating the JNK kinase SEK-1 (MKK4 and -7). Importantly, the MLK member MLK3/SPRK has been shown recently to be a direct target of ceramide and tumor necrosis factor-alpha (TNF-alpha) and to mediate the TNF-alpha and ceramide-induced JNK activation in Jurkat cells.

Activation of the Drosophila MLK by ceramide reveals TNF-alpha and ceramide as agonists of mammalian MLK3.

Mixed lineage kinases (MLKs) are MAPKKK members that activate JNK and reportedly lead to cell death. However, the agonist(s) that regulate MLK activity remain unknown. Here, we demonstrate ceramide as the activator of Drosophila MLK (dMLK) and identify ceramide and TNF-alpha as agonists of mammalian MLK3.