Detection and characterization of multiple forms of simian virus 40 large T antigen

Fanning, Ellen; Nowak, Barbara; Burger, Christa

doi:10.1128/jvi.37.1.92-102.1981

Cited by 130 publications

(97 citation statements)

References 40 publications

(47 reference statements)

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“…(We have not yet analyzed this peptide, because it represented a minor species.) Other investigators reported a positive correlation between the degree of phosphorylation of large T and its affinity for DNA (16,31); in addition, the DNA-binding capacity of large T was found to be restricted to its oligomeric forms (4), which are phosphorylated to a higher degree than monomers (10,16). On the other hand, Shaw and Tegtmeyer found no difference in the specific DNA-binding potential between phosphorylated and dephosphorylated large T (41).…”

Section: Discussionmentioning

confidence: 94%

“…Peptides containing identical phosphate acceptors but differing in their phosphorylation extent must derive from different molecular classes of large T. Several investigators described subspecies of large T differing in their aggregation state (4,10,16) or their DNA-binding capacity (16, 32) on one side, or their degree of phosphorylation on the other (15,31). It will be interesting to investigate how different aggregation forms or DNA-binding species of large T correlate with the phosphorylation state of specific sites.…”

Section: Discussionmentioning

confidence: 99%

“…How these different functions are exerted by a single protein is not understood, but it is possible that large T can assume different functional forms which are interconvertible by phosphor-ylation. Compatible with this possibility are the following findings: large T is phosphorylated at multiple sites (52) in a reversible fashion (9,38); subclasses of large T can be separated by isoelectric focusing (15); and correlations exist between the degree of phosphorylation of large T on one hand and its degree of oligomerization (10,16) or its affinity for DNA (31) on the other. However, the functional significance of these quantitative differences in the phosphorylation state is not yet clear.…”

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confidence: 99%

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Simian Virus 40 Large T Antigen Is Phosphorylated at Multiple Sites Clustered in Two Separate Regions

Scheidtmann

Echle

Walter

1982

J Virol

186

131

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The phosphorylation sites of simian virus 40 large T antigen were determined within the primary structure of the molecule. Exhaustive digestion of 32 P-labeled large T antigen with trypsin generated six major phosphopeptides which could be separated in a newly developed isobutyric acid-containing chromatography system. By partial tryptic digestion, large T antigen was cleaved into an amino-terminal fragment of 17,000 daltons and overlapping fragments from the carboxy-terminal region ranging in size between 71,000 and 13,000 daltons. The location of the phosphopeptides was then determined by fingerprint analyses of individual fragments. Their physical properties were analyzed by sizing on polyacrylamide gels and by sequential digestion and peptide mapping; their amino acid composition was determined by differential labeling with various amino acids. The amino-terminal 17,000-dalton fragment gave rise to only one phosphopeptide (phosphopeptide 3) that contained half of the phosphate label incorporated into large T antigen. It contained phosphoserine and phosphothreonine sites, all of which were clustered within a small segment between Cys 105 and Lys 127 . This segment contained five serines and two threonines. Among these, Ser 106 , Ser 123 , and Thr 124 were identified as phosphorylated residues; in addition, either one or both of Ser 111 and Ser 112 were phosphorylated. The neighboring residues, Ser 123 and Thr 124 , were found in three different phosphorylation states in that either Ser 123 or Thr 124 or both were phosphorylated. Phosphopeptides 1, 2, 4, 5, and 6 were all derived from a single fragment extending 26,000 daltons upstream from the carboxy terminus of large T antigen. Phosphopeptide 6 was identical with the previously determined phosphothreonine peptide phosphorylated at Thr 701 . Phosphopeptides 1, 2, 4, and 5 contained only serine-bound phosphate. Phosphopeptides 1, 2, and 4 represented overlapping peptides, all of which were phosphorylated at Ser 639 located next to a cluster of six acidic residues. In phosphopeptide 5, a large peptide ranging from Asn 653 to Arg 691 , at least two of seven serines were phosphorylated. Thus, large T antigen contains at least eight phosphorylation sites. Their clustering within two separate regions might correlate with structural and functional domains of this protein.

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Section: Discussionmentioning

confidence: 94%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Simian Virus 40 Large T Antigen Is Phosphorylated at Multiple Sites Clustered in Two Separate Regions

Scheidtmann

Echle

Walter

1982

J Virol

186

131

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“…The interactions of Nef with cellular factors are likely to be the key to the still enigmatic mechanisms of Nef function(s). We therefore tried to characterize these interactions in a general way by zone velocity sedimentation, an approach that has been successfully used to study interactions of simian virus 40 (SV40) T antigen [28,291. We report here an interaction of Nef with cellular components identifying actin as a major binding partner.…”

mentioning

confidence: 99%

Association of Human Immunodeficiency Virus Nef Protein with Actin is Myristoylation Dependent and Influences its Subcellular Localization

Fackler

Kienzle

Kremmer

et al. 1997

European Journal of Biochemistry

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Human immunodeficiency virus (HIV) Nef functions are thought to be mediated via interactions with cellular proteins. Utilizing zone velocity sedimentation in glycerol gradients we found that recombinant HIV‐1 Nef non‐covalently associates with actin forming a high‐molecular‐mass complex of 150–300 kDa. This Nef/actin complex was present in human B and T lymphocytes but not in insect cells and was dependent on the N‐terminal myristoylation of Nef, whereas the SH3‐binding proline motif of Nef was not involved. Despite being myristoylated, HIV‐2 Nef did not associate with actin. This might reflect differences in the subcellular localization of Nef since cell‐fractionation experiments revealed that HIV‐ 1Nef was virtually exclusively localized in the cytoskeletal (detergent‐insoluble) fraction whereas HIV‐ 2 Nef had significantly reduced affinity for the cytoskeleton. Colocalization experiments in HIV‐1‐in‐fected CD4+ fibroblasts revealed that Nef/actin complexes may also exist in HIV‐infected cells. This novel interaction of HIV‐1 Nef with actin provides insight into the association of Nef with cellular structures and reveals general differences in the interactions of the Nef proteins from HIV‐1 and HIV‐2.

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“…In vitro, large T binds to DNA, specifically to the origin of replication on the viral genome (20,48,54,58), and has ATPase activity, which may be an intrinsic property of the molecule (60). It exists in monomeric, dimeric, and tetrameric forms (4,11,39) and associates with a cellular 53,000-dalton (53K) protein (11,18). It is not understood how large T performs several distinct functions.…”

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confidence: 99%

DNA-binding activity of simian virus 40 large T antigen correlates with a distinct phosphorylation state

Scheidtmann¹,

Hardung²,

Echle³

et al. 1984

J Virol

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The state of phosphorylation and the relationship of various subclasses of simian virus 40 large T antigen (large T) differing in DNA-binding activity, degree of oligomerization, age, and subcellular distribution were investigated. Young large T (continuously labeled for 4 h late in infection) comprised about 20% of the total cellular large T. It was phosphorylated to a low degree and existed primarily in a monomeric form, sedimenting at 5S. More than 50% of this fraction bound to simian virus 40 DNA, preferentially to origincontaining sequences. Old large T (continuously labeled for 17 h, followed by a 4-h chase) represented the majority of the population. It was highly phosphorylated and predominantly in an oligomeric form, sedimenting at 15S to 23S. Only 10 to 20% of this fraction bound to simian virus 40 DNA. Another subclass of large T which was extracted from nuclei with 0.5 M salt resembled newly synthesized molecules in all properties tested; it was phosphorylated to a low degree, sedimented at SS, and bound to viral DNA with high efficiency (>70%). Two-dimensional phosphopeptide analysis of the individual subclasses revealed two distinct phosphorylation patterns, one characteristic for young, monomeric, and DNA-binding large T, the other for old, oligomeric, and non-DNA-binding large T. All sites previously identified in unfractionated large T (K. H. Scheidtmann et al., J. Virol. 44:116-133, 1982) were also phosphorylated in the various subclasses, but to different degrees. Peptide maps of the DNA-binding fraction, the 5S form, and the nuclear high-salt fraction showed two prominent phosphopeptides not previously characterized. Both peptides were derived from the amino-terminal region of large T, presumably involved in origin binding, and probably represent partially phosphorylated intermediates of known phosphopeptides. Our data show that the DNAbinding activity, age, and oligomerization of large T correlate with distinct states of phosphorylation. We propose that differential phosphorylation might play a role in the interaction of large T with DNA.

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Detection and characterization of multiple forms of simian virus 40 large T antigen

Cited by 130 publications

References 40 publications

Simian Virus 40 Large T Antigen Is Phosphorylated at Multiple Sites Clustered in Two Separate Regions

Simian Virus 40 Large T Antigen Is Phosphorylated at Multiple Sites Clustered in Two Separate Regions

Association of Human Immunodeficiency Virus Nef Protein with Actin is Myristoylation Dependent and Influences its Subcellular Localization

DNA-binding activity of simian virus 40 large T antigen correlates with a distinct phosphorylation state

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