Avian retrovirus nucleocapsid protein, pp12, produced in Escherichia coli has biochemical properties identical to unphosphorylated viral protein

Ra, Kyung-Soo; Xd, Fu; Am, Skalka; Leis, Jonathan

doi:10.1016/0378-1119(86)90340-9

Cited by 14 publications

(9 citation statements)

References 17 publications

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“…Although it has not been analyzed directly, we presume that the p32P°l protein expressed in bacteria is not phosphorylated (15). Thus, it was surprising to find that this protein migrated closer to the slower form of viral pp32 which Schiff and Grandgenett (27) have shown contains 2 to 3 phosphorylated residues.…”

Section: Discussionmentioning

confidence: 92%

Properties of avian sarcoma-leukosis virus pp32-related pol-endonucleases produced in Escherichia coli

Terry

Soltis

Katzman

et al. 1988

J Virol

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The gag-pol precursor protein of the avian sarcoma-leukosis virus is processed into three known pol-encoded mature polypeptides; the 95-and 63-kilodalton (kDa) and a subunits, respectively, of reverse transcriptase and the 32-kDa pp32 protein. The pp32 protein possesses DNA endonuclease activity and is produced from the precursor by two proteolytic cleavage events, one of which removes 4.1 kDa of protein from the C terminus. A 36-kDa protein (p36P°') which retains this C-terminal segment is detectable in small quantities in virions. We have constructed Escherichia coli plasmid clones that express the C-terminal domains of pol corresponding to pp32 and p36. These proteins have been purified by column chromatographic methods to near homogeneity. No significant differences could be detected in the enzymatic properties of the bacterially produced p32P"' and p36&"" proteins. Both possess DNA endonuclease activity and, like the pp32 protein isolated from virions, can cleave near the junction of two tandem avian sarcoma-leukosis virus long terminal repeats in double-stranded supercoiled DNA substrates. In the presence of Mg2+, both p32P"' and viral pp32 cleave either strand of DNA 2 nucleotides 5' to the junction.

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Section: Discussionmentioning

confidence: 92%

Properties of avian sarcoma-leukosis virus pp32-related pol-endonucleases produced in Escherichia coli

Terry

Soltis

Katzman

et al. 1988

J Virol

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“…GST‐Cdc42 was expressed in Escehrichia coli and purified on glutathione Sepharose 4‐B [29]. The NC protein from Rous sarcoma virus was expressed in E. coli and purified as described [30].…”

Section: Methodsmentioning

confidence: 99%

Substrates enhance autophosphorylation and activation of p21‐activated protein kinase γ‐PAK in the absence of activation loop phosphorylation

Jakobi

Huang

Walter

et al. 2000

European Journal of Biochemistry

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The p21‐activated protein kinase γ‐PAK from rabbit, expressed in insect cells, is activated following binding of Cdc42(GTPγS). The rate of autophosphorylation is increased fivefold and the protein kinase activity 13‐fold, as measured with the synthetic heptapeptide (AKRESAA). The mutant K278R, where the invariant lysine in the catalytic site is replaced by arginine, shows neither autophosphorylation nor activity. Replacement of the conserved threonine in the catalytic domain with alanine (T402A) reduces autophosphorylation and protein kinase activity to 1% that of the wild‐type γ‐PAK, indicating autophosphorylation of Thr402 in the activation loop is essential for protein kinase activity. In contrast, certain protein substrates such as histone 2B, histone 4 and myelin basic protein, stimulate both autophosphorylation and protein kinase activity to levels similar to those observed with Cdc42(GTPγS). This substrate‐level activation does not require autophosphorylation of Thr402 in the activation loop. As shown with T402A, the protein kinase activity with histone 4 is similar to that observed with recombinant wild‐type γ‐PAK. Basic proteins or peptides which are not substrates of γ‐PAK, such as histone 1 and polylysine, do not stimulate autophosphorylation or activity. Other substrates such as the Rous sarcoma virus protein NC are phosphorylated by γ‐PAK following activation by Cdc42(GTPγS), but are not phosphorylated by T402A. The data suggest that some substrates can override the requirement for Cdc42(GTPγS), by activating γ‐PAK directly.

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“…Site-directed oligonucleotide mutagenesis was carried out as described previously (16). All mutations were confirmed by nucleotide sequence analysis using the Sequenase method (2).…”

Section: Methodsmentioning

confidence: 99%

Genetic Selection for Balanced Retroviral Splicing: Novel Regulation Involving the Second Step Can Be Mediated by Transitions in the Polypyrimidine Tract

Bouck

Skalka

et al. 1995

Molecular and Cellular Biology

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Incomplete splicing is essential for retroviral replication; and in simple retroviruses, splicing regulation appears to occur entirely in cis. Our previous studies, using avian sarcoma virus, indicated that weak splicing signals allow transcripts to escape the splicing pathway. We also isolated a series of avian sarcoma virus mutants in which env mRNA splicing was regulated by mechanisms distinct from those of the wild-type virus. In vitro splicing experiments with one such mutant (insertion suppressor 1 [IS1]) revealed that exon 1 and lariat-exon 2 intermediates were produced (step 1) but the exons were not efficiently ligated (step 2). In this work, we have studied the mechanism of this second-step block as well as its biological relevance. Our results show that the second-step block can be overcome by extending the polypyrimidine tract, and this causes an oversplicing defect in vivo. The requirement for regulated splicing was exploited to isolate new suppressor mutations that restored viral growth by down-regulating splicing. One suppressor consisted of a single U-to-C transition in the polypyrimidine tract; a second included this same change as well as an additional U-to-C transition within a uridine stretch in the polypyrimidine tract. These suppressor mutations affected primarily the second step of splicing in vitro. These results support a specific role for the polypyrimidine tract in the second step of splicing and confirm that, in a biological system, uridines and cytosines are not functionally equivalent within the polypyrimidine tract. Unlike the wild-type virus, the second-step mutants displayed significant levels of lariat-exon 2 in vivo, suggesting a role for splicing intermediates in regulation. Our results indicate that splicing regulation can involve either the first or second step.Pre-mRNA splicing involves the accurate removal of introns and joining of exons (see references 14, 28, and 37 for reviews). In higher eukaryotes, these reactions require several degenerate cis-acting signals: the 5Ј splice site (AG/GURAGU), the 3Ј splice site (CAG/G), the branch point sequence (BPS; YNYU RAC), and the polypyrimidine tract (Y) n (Fig. 1A). These signal sequences are recognized by nuclear trans-acting factors: small nuclear ribonucleoprotein particles (snRNPs) and nonsnRNP protein factors. The U1, U2, U4, U5, and U6 snRNPs and other factors bind directly, or indirectly, to the pre-mRNA in an ordered manner, ultimately forming a large complex known as the spliceosome. The U1 snRNP initially recognizes the 5Ј splice site, and the U2 snRNP recognizes the BPS, both through RNA-RNA base pairing. The polypyrimidine tract is required for spliceosome formation, and one function of this element is to recruit a protein, U2AF, that stabilizes the binding of the U2 snRNP (21, 49). The polypyrimidine tract may also play an indirect role in selecting the 3Ј splice site through a scanning mechanism (40, 42). The functional strength of the polypyrimidine tract has been correlated with its length, pyrimidine content, a...

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Avian retrovirus nucleocapsid protein, pp12, produced in Escherichia coli has biochemical properties identical to unphosphorylated viral protein

Cited by 14 publications

References 17 publications

Properties of avian sarcoma-leukosis virus pp32-related pol-endonucleases produced in Escherichia coli

Properties of avian sarcoma-leukosis virus pp32-related pol-endonucleases produced in Escherichia coli

Substrates enhance autophosphorylation and activation of p21‐activated protein kinase γ‐PAK in the absence of activation loop phosphorylation

Genetic Selection for Balanced Retroviral Splicing: Novel Regulation Involving the Second Step Can Be Mediated by Transitions in the Polypyrimidine Tract

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