A protein kinase from wheat germ that phosphorylates the largest subunit of RNA polymerase II.

Guilfoyle, Tom J.

doi:10.1105/tpc.1.8.827

Cited by 29 publications

(8 citation statements)

References 35 publications

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“…The addition of λ-PPase reduced phosphorylation and increased the electrophoretic mobility of all CPKs during SDS/PAGE (Figure 3). The observed kDa mobility mass shifts were probably due to the removal of multiple phosphate groups; similar mobility shifts have been observed previously [43]. Recombinant 14-3-3χ (unphosphorylated) and ovalbumin (a known phosphoprotein) were included as negative and positive controls respectively.…”

Section: Resultssupporting

confidence: 80%

Multisite phosphorylation of 14-3-3 proteins by calcium-dependent protein kinases

Swatek

Wilson

Ahsan

et al. 2014

Biochemical Journal

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Plant 14-3-3 proteins are phosphorylated at multiple sites in vivo; however, the protein kinase(s) responsible are unknown. Of the 34 CPK (calcium-dependent protein kinase) paralogues in Arabidopsis thaliana, three (CPK1, CPK24 and CPK28) contain a canonical 14-3-3-binding motif. These three, in addition to CPK3, CPK6 and CPK8, were tested for activity against recombinant 14-3-3 proteins χ and ε. Using an MS-based quantitative assay we demonstrate phosphorylation of 14-3-3 χ and ε at a total of seven sites, one of which is an in vivo site discovered in Arabidopsis. CPK autophosphorylation was also comprehensively monitored by MS and revealed a total of 45 sites among the six CPKs analysed, most of which were located within the N-terminal variable and catalytic domains. Among these CPK autophosphorylation sites was Tyr463 within the calcium-binding EF-hand domain of CPK28. Of all CPKs assayed, CPK28, which contained an autophosphorylation site (Ser43) within a canonical 14-3-3-binding motif, showed the highest activity against 14-3-3 proteins. Phosphomimetic mutagenesis of Ser72 to aspartate on 14-3-3χ, which is adjacent to the 14-3-3-binding cleft and conserved among all 14-3-3 isoforms, prevented 14-3-3-mediated inhibition of phosphorylated nitrate reductase.

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Section: Resultssupporting

confidence: 80%

Multisite phosphorylation of 14-3-3 proteins by calcium-dependent protein kinases

Swatek

Wilson

Ahsan

et al. 2014

Biochemical Journal

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“…The total NTP concentration affected the size of the runoff transcript produced from the gliadin promoter; 20 #M was optimal; lower concentrations (<5/~M) produced less overall activity while higher concentrations (> 100/~M) resulted in the synthesis of larger transcripts than expected for correct initiation from the gliadin promoter. This observation suggests that precise selection of the initiation sites may be influenced directly by the NTP concentration, or indirectly by some other nucleotide-dependent activity [8,14], acting on some component of the initiation apparatus. The effect of NTP concentration is being studied further but optimum conditions should be empirically determined on each promoter being tested since we note that the size of the transcript produced from pHc35S-pA was not affected by NTP concentration.…”

Section: Optimal Reaction Conditions Of the Rice In Vitro Transcriptimentioning

confidence: 99%

“…The shaded regions represent linker sequences carried over in subcloning steps. B. pHc-35S-pA; a 460 bp segment of the 35S promoter from the Hinc II site at position 7014 [12] (-419 relative to the major start site mapped in vivo [14] to the Barn HI site of pBIl21 [15] was fused upstream of the same poly(dA) containing cDNA fragment shown for pGlia-pA. C. Nucleotide sequence of the 250 bp gliadin promoter element of pGlia-pA. The CCAAT and TATA elements are enclosed in boxes.…”

Section: Promoter Elements and In Vitro Transcription Assaymentioning

confidence: 99%

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Accurate in vitro transcription of plant promoters with nuclear extracts prepared from cultured plant cells

Roberts

Okita

1991

Plant Mol Biol

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A simple method is presented for the preparation of nuclear extracts from suspension cultures of rice, wheat and tobacco cells. These extracts are shown to be capable of RNA Polymerase II-dependent transcription from two plant promoters in vitro; a 250 bp fragment of a wheat gliadin promoter containing sequences from -167 bp to +83 relative to the in vivo transcriptional initiation site and two fragments of the CaMV 35S promoter, containing sequences from -419 to +17, and from -90 to +17. Using the rice extract, transcription is shown to be extract-dependent, DNA-dependent, alpha-amanatin-sensitive, promoter-dependent, and accurate with respect to initiation site selection on the gliadin promoter and the -90 to +17 35S promoter, but not accurate on the -419 to +17 35S promoter.

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“…In addition, several laboratories have characterized protein kinases capable of specifically and extensively phosphorylating the CTD [ 17,29,43]. We have determined the nucleotide sequence of the single-copy gene RPB1 and two partial cDNA clones from Arabidopsis, as well as several genomic and partial cDNA clones from soybean.…”

Section: Introductionmentioning

confidence: 99%

Analysis of the genes encoding the largest subunit of RNA polymerase II in Arabidopsis and soybean

1990

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We have cloned and sequenced the gene encoding the largest subunit of RNA polymerase II (RPB1) from Arabidopsis thaliana and partially sequenced genes from soybean (Glycine max). We have also determined the nucleotide sequence for a number of cDNA clones which encode the carboxyl terminal domains (CTDs) of RNA polymerase II from both soybean and Arabidopsis. The Arabidopsis RPB1 gene encodes a polypeptide of approximately 205 kDa, consists of 12 exons, and encompasses more than 8 kb. Predicted amino acid sequence shows eight regions of similarity with the largest subunit of other prokaryotic and eukaryotic RNA polymerases, as well as a highly conserved CTD unique to RNA polymerase II. The CTDs in plants, like those in most other eukaryotes, consist of tandem heptapeptide repeats with the consensus amino acid sequence PTSPSYS. The portion of RPB1 which encodes the CTD in plants differs from that of RPB1 of animals and lower eukaryotes. All the plant genes examined contain 2-3 introns within the CTD encoding regions, and at least two plant genes contain an alternatively spliced intron in the 3' untranslated region. Several clustered amino acid substitutions in the CTD are conserved in the two plant species examined, but are not found in other eukaryotes. RPB1 is encoded by a multigene family in soybean, but a single gene encodes this subunit in Arabidopsis and most other eukaryotes.

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A protein kinase from wheat germ that phosphorylates the largest subunit of RNA polymerase II.

Cited by 29 publications

References 35 publications

Multisite phosphorylation of 14-3-3 proteins by calcium-dependent protein kinases

Multisite phosphorylation of 14-3-3 proteins by calcium-dependent protein kinases

Accurate in vitro transcription of plant promoters with nuclear extracts prepared from cultured plant cells

Analysis of the genes encoding the largest subunit of RNA polymerase II in Arabidopsis and soybean

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