Targeted Proteomic Analysis of 14-3-3ς, a p53 Effector Commonly Silenced in Cancer

Molecular Oncology, Max-Planck-Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried/Munich, Germany.
Molecular &amp Cellular Proteomics (Impact Factor: 7.25). 07/2005; 4(6):785-95. DOI: 10.1074/mcp.M500021-MCP200
Source: PubMed

ABSTRACT To comprehensively identify proteins interacting with 14-3-3 sigma in vivo, tandem affinity purification and the multidimensional protein identification technology were combined to characterize 117 proteins associated with 14-3-3 sigma in human cells. The majority of identified proteins contained one or several phosphorylatable 14-3-3-binding sites indicating a potential direct interaction with 14-3-3 sigma. 25 proteins were not previously assigned to any function and were named SIP2-26 (for 14-3-3 sigma-interacting protein). Among the 92 interactors with known function were a number of proteins previously implicated in oncogenic signaling (APC, A-RAF, B-RAF, and c-RAF) and cell cycle regulation (AJUBA, c-TAK, PTOV-1, and WEE1). The largest functional classes comprised proteins involved in the regulation of cytoskeletal dynamics, polarity, adhesion, mitogenic signaling, and motility. Accordingly ectopic 14-3-3 sigma expression prevented cellular migration in a wounding assay and enhanced mitogen-activated protein kinase signaling. The functional diversity of the identified proteins indicates that induction of 14-3-3 sigma could allow p53 to affect numerous processes in addition to the previously characterized inhibitory effect on G2/M progression. The data suggest that the cancer-specific loss of 14-3-3 sigma expression by epigenetic silencing or p53 mutations contributes to cancer formation by multiple routes.

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    • "Significant efforts Table 1. Common 14-3-3ζ-interacting proteins 14-3-3 zeta proteomics 79 Int J Physiol Pathophysiol Pharmacol 2012;4(2):74-83 have been expended toward full characterization of the 14-3-3 interactome using proteomics -based approaches [9] and the present work extends findings on other isoforms [12] [13]. Here, we used recombinant human Histagged 14-3-3ζ to elute 14- 3-3 binding proteins from mouse hippocampus. "
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    ABSTRACT: 14-3-3 proteins are ubiquitous molecular chaperones with important roles in brain development and neuronal function. Altered expression of 14-3-3 proteins has been reported in several neurologic and neurodegenerative disorders and identifying 14-3-3 binding proteins may provide important insights into the physiologic and pathophysiologic roles of these proteins. Particular interest has emerged on 14-3-3 zeta (ζ) in the setting of neuronal injury because reducing 14-3-3ζ levels triggers an endoplasmic reticulum stress-like response in neurons and increases vulnerability to excitotoxicity. Here we examined the subcellular distribution of 14-3-3ζ in the mouse hippocampus. We then used recombinant His-tagged 14-3-3ζ to pull-down interacting proteins from the mouse hippocampus followed by identification by liquid chromatography-mass spectrometry. 14-3-3ζ protein was present in the cytoplasm, microsomal compartment, nucleus and mitochondrial fractions of the mouse hippocampus. Recombinant 14-3-3ζ eluted 13 known 14-3-3 binding partners, including three other 14-3-3 isoforms, and 16 other proteins which have not previously been reported to bind 14-3-3ζ. The present study identifies potentially novel 14-3-3ζ binding proteins and contributes to defining the 14-3-3ζ interactome in the mouse brain.
    International Journal of Physiology, Pathophysiology and Pharmacology 01/2012; 4(2):74-83.
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    • "Affinity purification of cellular 14-3-3 binding proteins in proteomic studies provide evidence for several hundred different binding partners (possibly >500) associated with most cellular processes [1] [2] [3] [4] [5] [6] [7] [8]. Although the associations with many of these binding partners have not been completely verified yet, these proteomic and interactomic studies clearly illustrate the diverse biological functions associated with this protein family. "
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    ABSTRACT: Thirty years ago, it was discovered that 14-3-3 proteins could activate enzymes involved in amino acid metabolism. In the following decades, 14-3-3s have been shown to be involved in many different signaling pathways that modulate cellular and whole body energy and nutrient homeostasis. Large scale screening for cellular binding partners of 14-3-3 has identified numerous proteins that participate in regulation of metabolic pathways, although only a minority of these targets have yet been subject to detailed studies. Because of the wide distribution of potential 14-3-3 targets and the resurging interest in metabolic pathway control in diseases like cancer, diabetes, obesity and cardiovascular disease, we review the role of 14-3-3 proteins in the regulation of core and specialized cellular metabolic functions. We cite illustrative examples of 14-3-3 action through their direct modulation of individual enzymes and through regulation of master switches in cellular pathways, such as insulin signaling, mTOR- and AMP dependent kinase signaling pathways, as well as regulation of autophagy. We further illustrate the quantitative impact of 14-3-3 association on signal response at the target protein level and we discuss implications of recent findings showing 14-3-3 protein membrane binding of target proteins.
    Seminars in Cell and Developmental Biology 08/2011; 22(7):713-9. DOI:10.1016/j.semcdb.2011.08.008 · 5.97 Impact Factor
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    • "A reduced capacity in filling the gap was observed in keratinocyte monolayers from transgenic mice as compared with wild-type ones. As this capacity depends on both proliferation and migration, and 14-3-3s was found to interact with several proteins involved in cell motility (Benzinger et al., 2005), keratinocyte migration was also investigated using a Boyden chamber assay. This revealed that keratinocytes overexpressing 14-3-3s manifest significantly reduced motility (Figure 4f). "
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    ABSTRACT: The 14-3-3 protein family controls diverse biochemical processes through interaction with phosphorylated consensus sequences in protein targets. Its epithelial specific member, 14-3-3σ, also known as stratifin, is highly expressed in differentiated keratinocytes, and in vitro evidence indicates that 14-3-3σ downregulation leads to keratinocyte immortalization. To define the role of 14-3-3σ in skin homeostasis in vivo, we generated transgenic mice overexpressing 14-3-3σ in proliferating keratinocytes of the epidermis and hair follicle. Transgenic animals show decreased epidermal thickness and hair follicle density associated with reduced number of proliferating keratinocytes and decreased levels of keratins 14, 5, and 15. Primary keratinocytes isolated from transgenic mice manifest reduced proliferation and migration. Moreover, clonogenicity assessment and label-retaining analysis reveal a reduction in keratinocyte progenitor cell number in transgenic mice. Response to IGF-1 is strongly impaired in cultured transgenic keratinocytes compared with wild-type cells. Consistently, activation of phosphoinositol 3-kinase (PI3K), AKT, and Rac1, all IGF-1 downstream mediators, is reduced. Our results demonstrate that 14-3-3σ controls the in vivo epidermal proliferation-differentiation switch by reducing proliferative potential and forcing keratinocytes to exit the cell cycle, and that this effect associates with inhibition of the IGF-1 pathway.
    Journal of Investigative Dermatology 06/2011; 131(9):1821-9. DOI:10.1038/jid.2011.137 · 6.37 Impact Factor
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