Mass spectrometric characterization of stathmin isoforms separated by 2D PAGE

Müller, Dieter; Schindler, Patrick; Coulot, M.; Voshol, Hans; Oostrum, Jan van

doi:10.1002/(sici)1096-9888(199904)34:4<336::aid-jms765>3.0.co;2-u

Cited by 52 publications

(46 citation statements)

References 22 publications

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“…Confirmation of the assignment of the N-terminal sequences was performed by nano-ESMS/MS sequencing. Direct selection of the triply charged parent ion at m/z 421.22 from the Lys-C digest of the induced 14-3-3␥ isoform and subsequent fragmentation produced sufficient y and b type fragment ions to identify unambiguously the sequence of the peptide as M(oxi)-VDREQLVQK (1)(2)(3)(4)(5)(6)(7)(8)(9)(10). A similar analysis of the aminoterminal peptide of the tryptic digest of the constitutive 14-3-3␥ confirmed the complete sequence as acetyl-VDREQLVQK (2-10) (data not shown).…”

Section: A Proteomics Approach Identifies An Alteration In a 14-3-3mentioning

confidence: 99%

Proteomics-based Target Identification

Towbin

Bair

DeCaprio

et al. 2003

Journal of Biological Chemistry

143

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LAF389 is a synthetic analogue of bengamides, a class of marine natural products that produce inhibitory effects on tumor growth in vitro and in vivo. A proteomicsbased approach has been used to identify signaling pathways affected by bengamides. LAF389 treatment of cells resulted in altered mobility of a subset of proteins on two-dimensional gel electrophoresis. Detailed analysis of one of the proteins, 14-3-3␥, showed that bengamide treatment resulted in retention of the amino-terminal methionine, suggesting that bengamides directly or indirectly inhibited methionine aminopeptidases (MetAps). Both known MetAps are inhibited by LAF389. Short interfering RNA suppression of MetAp2 also altered amino-terminal processing of 14-3-3␥. A high resolution structure of human MetAp2 co-crystallized with a bengamide shows that the compound binds in a manner that mimics peptide substrates. Additionally, the structure reveals that three key hydroxyl groups on the inhibitor coordinate the di-cobalt center in the enzyme active site.

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Section: A Proteomics Approach Identifies An Alteration In a 14-3-3mentioning

confidence: 99%

Proteomics-based Target Identification

Towbin

Bair

DeCaprio

et al. 2003

Journal of Biological Chemistry

143

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“…The two major phosphorylated isoforms Op18a and Op18b have been previously reported as being phosphorylated at Ser-25 for Op18a and Ser-25 and Ser-38 for Op18b (26 -29). The pattern of Op18 expression among these isoforms in lung adenocarcinomas is different from that observed in acute leukemia, which expresses three isoforms (12), and a pre-B 697 line, which expresses seven isoforms (38). In the lung tumors examined in the present study, the abundance of the unphosphorylated Op18 isoform was much higher than the abundance of the other three phos- phorylated forms, yet a strong correlation was observed between the expression of all forms, indicating that the subsequent modification of the phosphorylated isoforms is directly related to the overall level of the unphosphorylated form.…”

Section: Op18 Expression In Lung Adenocarcinomasmentioning

confidence: 99%

“…For example, transcription factors (E2F, AP-2, and Sp1) can increase Op18 expression (38), whereas p53 appears to repress the Op18 expression (43). Protein kinases such as cyclin-dependent kinases (CDKs), the cAMP-dependent protein kinase, the mitogen-activated protein/extracellular signal-regulated kinase family, and the Ca 2ϩ /calmodulin-dependent kinase IV/Gr can cause extensive phosphorylation of Op18 protein indicating that Op18 can be influenced by a number of signal transduction pathways (10).…”

Section: Op18 Expression In Lung Adenocarcinomasmentioning

confidence: 99%

Overexpression of Oncoprotein 18 Correlates with Poor Differentiation in Lung Adenocarcinomas

Chen

Wang

Gharib

et al. 2003

Molecular & Cellular Proteomics

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We examined the expression of oncoprotein 18 (Op18) in 93 lung adenocarcinomas and 10 uninvolved lung samples using quantitative two-dimensional PAGE analysis with confirmation by mass spectrometry and two-dimensional Western blot analysis. mRNA expression was examined using oligonucleotide microarrays, and the cellular localization of the Op18 protein was examined using immunohistochemical analysis of tissue microarrays. Three phosphorylated forms and one unphosphorylated form of the Op18 protein were identified and found to be overexpressed in lung adenocarcinomas as compared with normal lung. The percentage of phosphorylated to total Op18 protein isoforms increased from 3.2% in normal lung to 7.9% in lung tumors. Both the phosphorylated and unphosphorylated Op18 proteins were significantly increased in poorly differentiated tumors as compared with moderately or well differentiated lung adenocarcinomas (p < 0.03), suggesting that up-regulated expression of Op18 reflects a poor differentiation status and higher cell proliferation rates. This was further verified in A549 and SKLU1 lung adenocarcinoma cell lines by examining Op18 levels and phosphorylation status following treatment that altered either cell proliferation or differentiation. The increased expression of Op18 protein was significantly correlated with its mRNA level indicating that increased transcription likely underlies elevated expression of Op18. The overexpression of Op18 proteins in poorly differentiated lung adenocarcinomas and the elevated expression of the phosphorylated forms of

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“…Interestingly, phosphorylation of stathmin at Ser 38 is thought to be via cyclin-dependent kinases during mitosis and results in reduced microtubule-destabilizing activity of stathmin (5,35,37). There are at least 14 isoforms of stathmin that migrate on 2-DE gels, two unphosphorylated and 12 increasingly phosphorylated proteins (48,71), and it has been suggested that regulation of microtubule dynamics by stathmin phosphorylation could be involved in fundamental processes associated with the reorganization of the cytoskeleton, such as neuronal differentiation or synaptic plasticity. The observation that desacetyl ␣-MSH increased the Ser 38 -phosphorylated isoform of stathmin suggests that desacetyl ␣-MSH may reduce the microtubule destabilizing effect and oppose the action of ␣-MSH, which increases stathmin expression but not phosphorylation.…”

Section: Discussionmentioning

confidence: 99%

Peripherally administered desacetyl α-MSH and α-MSH both influence postnatal rat growth and associated rat hypothalamic protein expression

Wu¹,

Greenwood²,

Cooney³

et al. 2006

American Journal of Physiology-Endocrinology and Metabolism

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. Peripherally administered desacetyl ␣-MSH and ␣-MSH both influence postnatal rat growth and associated rat hypothalamic protein expression. Am J Physiol Endocrinol Metab 291: E1372-E1380, 2006. First published July 25, 2006 doi:10.1152/ajpendo.00480.2005.-Desacetyl ␣-MSH predominates over ␣-MSH during development, but whether it is biologically active and has a physiological role is unclear. We compared the effects of 0.3␣-MSH on postnatal body growth by administering the peptides subcutaneously daily for postnatal days 0 -14 and also used a twodimensional gel electrophoresis gel-based proteomic approach to analyze protein changes in hypothalami, the relay center for body weight and growth regulation, after 14 days of treatment. We found that the growth rate between days 1 and 10 was significantly decreased by desacetyl ␣-MSH but not by ␣-MSH, but by day 14, a time reported for development of a mature pattern of hypothalamic innervation, both peptides had significantly increased neonatal growth compared with PBS-treated control rats. Desacetyl ␣-MSH significantly increased spleen weight, but ␣-MSH had no effect. ␣-MSH significantly decreased kidney weight, but desacetyl ␣-MSH had no effect. Both desacetyl ␣-MSH and ␣-MSH significantly decreased brain weight. By 14 days, both peptides significantly changed expression of a number of hypothalamic proteins, specifically metabolic enzymes, cytoskeleton, signaling, and stress response proteins. We show that peripherally administered desacetyl ␣-MSH is biologically active and induces responses that can differ from those for ␣-MSH. In conclusion, desacetyl ␣-MSH appears to be an important regulator of neonatal rat growth. melanocortin peptides; ␣-melanocyte-stimulating hormone; melanocortin receptors; proopiomelanocortin; hypothalamus THE PIVOTAL ROLE of the melanocortin system in regulation of apetite, metabolism, body size, and body weight is demonstrated clearly by the human melanocortin-4 receptor (MC4R) (24, 65) and proopiomelanocortin (POMC) (34) variants, three knockout mouse models (POMC, MC3R, and MC4R) (10,14,29,70), the spontaneously occurring dominant agouti mouse (68), and a mouse ectopically overexpressing agouti generelated peptide (26), all of which promote obesity. Still unresolved, though, are the roles of each POMC-derived peptide and the central melanocortin-signaling pathways.The melanocortin peptides ␣-melanocyte-stimulating hormone (␣-MSH) and desacetyl ␣-MSH are two endogenous peptides derived from a precursor protein, POMC, through posttranslational processing (46). NH 2 -terminal acetylation of desacetyl ␣-MSH to form ␣-MSH occurs in secretory vesicles just prior to exocytosis (42, 66), but not all desacetyl ␣-MSH is acetylated, since desacetyl ␣-MSH is present in the circulation and brain of rodents and humans. The major form of plasma immunoreactive MSH is ␣-MSH in rodents and desacetyl ␣-MSH in humans (22,40), and the relative abundance of these two MSH peptides is developmentally regulated (11,23). Desacetyl ␣-MSH predominates in rodent ...

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Mass spectrometric characterization of stathmin isoforms separated by 2D PAGE

Cited by 52 publications

References 22 publications

Proteomics-based Target Identification

Proteomics-based Target Identification

Overexpression of Oncoprotein 18 Correlates with Poor Differentiation in Lung Adenocarcinomas

Peripherally administered desacetyl α-MSH and α-MSH both influence postnatal rat growth and associated rat hypothalamic protein expression

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