High‐Sensitivity sequencing of large proteins: Partial structure of the rapamycin‐fkbp12 target

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MEF, a recently identified member of the E74 family of ETS-related transcription factors, is a strong transcriptional activator of cytokine gene expression. Using a green fluorescent protein gene reporter plasmid regulated by an MEF-responsive promoter, we determined that the transcriptional activity of MEF is largely restricted to the G 1 phase of the cell cycle. MEF-dependent transcription was suppressed by the expression of cyclin A but not by cyclin D or cyclin E. This effect was due to the kinase activity generated by cyclin A expression, as co-expression of the cyclin-dependent kinase inhibitors p21 or p27, or a dominant negative form of CDK2 (DNK2), abrogated the reduction of MEF transcriptional activity by cyclin A. Cyclin A-CDK2 phosphorylated MEF protein in vitro more efficiently than cyclin D-CDK4 or cyclin E-CDK2, and phosphorylation of MEF by cyclin A-CDK2 reduced its ability to bind DNA. We determined one site of phosphorylation by cyclin A-CDK2 at the C terminus of MEF, using mass-spectrometry; mutation of three serine or threonine residues in this region significantly reduced phosphorylation of MEF by cyclin A and reduced cyclin A-mediated suppression of its transactivating activity. These amino acid substitutions also reduced the restriction of MEF activity to G 1 . Phosphorylation of MEF by the cyclin A-CDK2 complex controls its transcriptional activity during the cell cycle, establishing a novel link between the ETS family of proteins and the cell cycle machinery.Critical events occur during the G 1 phase of the cell cycle that determine whether a cell will continue proliferating or will withdraw from the cell cycle and undergo differentiation or apoptosis (1). Transcription factors play a key role in these events, and their activity can be modified during G 1 by a variety of intracellular and extracellular signals that alter their amount or their ability to interact with other proteins or with DNA (2).Cyclin-dependent kinases (CDKs) 1 play a critical role in the commitment of a cell to proliferate; commitment occurs during the G 1 to S transition, at a time called the restriction point. CDKs are activated by their association with regulatory subunits known as cyclins. The transition from G 1 to S requires the temporal activation of cyclin D-CDK4 or cyclin D-CDK6, cyclin E-CDK2, and cyclin A-CDK2, while cyclin B-CDK1 (CDC2) is essential for the G 2 /M transition (3). Cyclin D-and cyclin E-dependent kinases regulate the activity of the transcription factor E2F, which controls the expression of a variety of genes necessary for DNA synthesis (4, 5). Cyclin-CDK complexes phosphorylate the retinoblastoma gene product (Rb), releasing E2F from its sequestration by Rb and allowing E2F to transactivate genes essential for S phase, such as DNA polymerase-␣, thymidine kinase, and cyclins E and A. Both cyclin E-CDK2 and cyclin A-CDK2 can form quaternary complexes with the Rb-related protein p107 and with E2F during S phase; the exact function of these complexes remains obscure (6, 7).The ETS family of tra...

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Cyclin A-dependent Phosphorylation of the ETS-related Protein, MEF, Restricts Its Activity to the G1 Phase of the Cell Cycle

Miyazaki¹,

Boccuni²,

Mao³

et al. 2001

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“…Protein Sequence Analysis-Internal amino acid sequence analysis was performed as described (24,25). Total cell lysates were separated on preparative two-dimensional gels and transferred to nitrocellulose membranes (Schleicher & Schull) using a large format (38 ϫ 46 ϫ 20 cm) electroblotter (Polytech Inc., Somerville, MA), which allowed for as many as six two-dimensional gels to be transferred simultaneously (26,27).…”

Section: Methodsmentioning

confidence: 99%

Components of the Protein Synthesis and Folding Machinery Are Induced in Vascular Smooth Muscle Cells by Hypertrophic and Hyperplastic Agents

Patton

Erdjument‐Bromage

Marks

et al. 1995

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Vascular smooth muscle cells (VSMC) are the principal cellular component of the blood vessel wall. Atherosclerosis, hypertension, and angiogenesis are associated with abnormal VSMC growth. Angiotensin II is hypertrophic for cultured adult rat aortic VSMC, whereas platelet-derived growth factor and serum are hyperplastic. To identify changes in specific proteins associated with either hyperplastic or hypertrophic growth, high resolution two-dimensional gel electrophoresis was performed on extracts from quiescent rat aortic VSMC and from VSMC exposed for 24 h to growth factors (10% fetal calf serum, platelet-derived growth factor, or angiotensin II). 12 proteins were up-regulated and 5 down-regulated by treatment with growth factors. Eight of the up-regulated and one of the down-regulated proteins were identified by internal protein microsequencing from electroblotted two-dimensional gels or by co-electrophoresis of purified proteins in two-dimensional gels. Four of the proteins up-regulated by growth factors were identified as mediators of protein folding. These were heat shock proteins, HSP-60 and HSP-70, protein disulfide isomerase, and protein disulfide isomerase isozyme Q-2. Additional proteins were identified as elongation factor EF-1␤, a component of the protein synthesis apparatus, and calreticulin, another putative molecular chaperone. Vimentin and actin were also upregulated, whereas an isoform of myosin heavy chain was down-regulated. Hyperplastic and hypertrophic growth were accompanied by similar changes in protein expression, suggesting that both types of growth require up-regulation of the protein synthesis and folding machinery.Growth and migration of VSMC 1 are considered to be key events in the pathogenesis of atherosclerosis, hypertension, and angiogenesis (1). VSMC display two distinct growth responses: hyperplasia, characterized by increased DNA and protein synthesis as well as cell division, and hypertrophy, characterized by increased cell size and protein content without DNA synthesis or cell division (2). Atherogenesis is characterized by the hyperplastic response, involving the migration of VSMC from the vessel media to the intima and the proliferation of medial and intimal VSMC (1). Chronic hypertension involves predominantly enlargement of preexisting VSMC (hypertrophy) within the media of the blood vessel as well as proliferation (2). In cell culture, the nature of the growth stimulus, rather than intrinsic differences in growth responsiveness of distinct cell subpopulations, appears to determine whether VSMC undergo hyperplasia or hypertrophy (2). PDGF and serum mediate a hyperplastic response in adult rat aortic VSMC (3). In the same cells, Ang, arginine vasopressin, and thrombin more typically mediate a hypertrophic response (3-9).Considerable progress has been made in elucidating the molecular events associated with hypertrophic and hyperplastic VSMC growth. Many studies have focused on examining mRNAs induced by growth factor activation of VSMC. These have identified mRNAs encoding...

“…Briefly, membrane bound proteins were digested in situ with trypsin, and the resulting peptides were separated by narrowbore reverse phase high pressure liquid chromatography. Selected peptides were then subjected to chemical microsequencing and matrix-assisted laser desorption ionization mass spectrometry, also as described previously (42,43). The 14-kDa band (p14) in the Sed5p immunoprecipitate from extracts of sec18 -1 at the nonpermissive temperature was identified by 2 The World Wide Web address is http://ulrec3.unil.ch.…”

Section: Yeast Strains and Media-yeast Strainsmentioning

confidence: 99%

Ykt6p, a Prenylated SNARE Essential for Endoplasmic Reticulum-Golgi Transport

McNew¹,

Søgaard²,

Lampen³

et al. 1997

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222

220

Vesicular transport between secretory compartments requires specific recognition molecules called SNAREs. Here we report the identification of three putative SNAREs, p14 (Sft1p), p28 (Gos1p), and a detailed characterization of p26 (Ykt6p). All three were originally isolated as interacting partners of the cis Golgi target membrane-associated SNARE Sed5p, when Sec18p (yeast NSF) was inactivated. YKT6 is an essential gene that codes for a novel vesicle-associated SNARE functioning at the endoplasmic reticulum-Golgi transport step in the yeast secretory pathway. Depletion of Ykt6p results in the accumulation of the p1 precursor (endoplasmic reticulum form) of the vacuolar enzyme carboxypeptidase Y and morphological abnormalities consistent with a defect in secretion. Membrane localization of Ykt6p is essential for protein function and is normally mediated by isoprenylation. However, replacement of the isoprenylation motif with a bona fide transmembrane anchor results in a functional protein confirming that membrane localization, but not isoprenylation per se, is required for function. Ykt6p and its homologues are highly conserved from yeast to human as demonstrated by the functional complementation of the loss of Ykt6p by its human counterpart. This is the first example of a human SNARE protein functionally replacing a yeast SNARE. This observation implies that the specific details of the vesicle targeting code, like the genetic code, are conserved in evolution.