Evidence for new phosphorylation sites for protein kinase C and cyclic AMP‐dependent protein kinase in bovine heart 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase

Rider, Mark H.; Damme, Josef Van; Vertommen, Didier; Michel, Alain; Vandekerckhove, Joël; Hue, Louis

doi:10.1016/0014-5793(92)81315-d

Cited by 26 publications

(29 citation statements)

References 13 publications

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“…Thus, the H54 isoform lacks all these sites although it still shares with the H59, H60 and H61 isoforms SerR4, which is phosphorylated by PKC [7]. The difference in C-terminal sequences also bears on the overall subunit charge (see above), which is liable to change upon phosphorylation.…”

Section: Discussionmentioning

confidence: 99%

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Cloning and expression of novel isoforms of 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase from bovine heart

Vidal¹,

Crepin²,

Rider³

et al. 1993

FEBS Letters

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Distinct 6‐phosphofructo‐2‐kinase (PFK‐2)/fructose 2,6‐bisphosphatase (FBPase‐2) cDNAs were cloned from bovine heart, showing that PFK‐2/FBPase‐2 gene B, which contains 16 exons, codes for at least five mRNAs. Three of them (B1, B2, B4) could encode the 58,000‐M r isozyme. In B2 mRNA, exon 15 encodes four more residues than in Bl. In B4 mRNA, exon 15 encodes six more residues than in B1, butexon 16 (20 residues) is missing. B3 mRNA corresponds to the 54,000‐M r isozyme. It lacks exon 15 and also differs from the other mRNAs in the 5' noncoding region. B5 mRNA encodes a truncated form. When expressed in E. coli, the recombinant isoforms corresponding to all these mRNAs except B5 exhibited PFK‐2 activity.

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Section: Discussionmentioning

confidence: 99%

“…Indeed, exon 15 codes for Ser466 which is phosphorylated by PKA and PKC, for Thr4" which is phosphorylated by PKC, and for Ser483 which is phosphorylated by PKA [7,8,11]. Thus, the H54 isoform lacks all these sites although it still shares with the H59, H60 and H61 isoforms SerR4, which is phosphorylated by PKC [7].…”

Section: Discussionmentioning

confidence: 99%

Cloning and expression of novel isoforms of 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase from bovine heart

Vidal¹,

Crepin²,

Rider³

et al. 1993

FEBS Letters

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“…Phosphopeptides were identified in MS 2 mode by the loss of H 3 PO 4 (98 Da) under low collision-induced dissociation energy, and the phosphorylated residue was pinpointed in MS 3 mode. In some cases, radioactive peaks were further purified for Edman sequencing (30).…”

Section: Methodsmentioning

confidence: 99%

Identification of Phosphorylation Sites in AMP-activated Protein Kinase (AMPK) for Upstream AMPK Kinases and Study of Their Roles by Site-directed Mutagenesis

Woods

Vertommen

Neumann

et al. 2003

Journal of Biological Chemistry

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213

187

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Bacterially expressed heterotrimeric (␣ 1 , ␤ 1 , and ␥ 1 ) wild-type, catalytically inactive, and constitutively active forms of AMP-activated protein kinase (AMPK) were used to study phosphorylation by an upstream AMPK kinase preparation. Here, we report the identification of two new phosphorylation sites in the ␣-subunit, viz. were not. Transfection of the non-phosphorylatable S485A and T258A mutants in CCL13 cells subjected to stresses known to activate AMPK either by increasing the AMP:ATP ratio (slow lysis) or without changing adenine nucleotide concentrations (hyperosmolarity) resulted in no significant differences in AMPK activation. All three sites within the ␣-subunit were phosphorylated in vivo, as seen in AMPK immunoprecipitated from anoxic rat liver. In transfected CCL13 cells, the level of Ser 485 phosphorylation did not change upon AMPK activation. The newly identified phosphorylation sites could play a subtle role in the regulation of AMPK, e.g. in subcellular localization or substrate recognition.

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“…The final incubation volumes were 0.15 ml for PKA, p70 s6k , and MAPKAP kinase-1 and 0.3 ml for PKB. After 2 h at 30°C, protein was precipitated (17) and digested in 0.2 ml of 0.1 M Tris-Cl, pH 8.6, and 2 M urea with 1 g of bovine trypsin overnight at 30°C. Peptides were separated by narrow bore HPLC (15), collected by hand in Eppendorf tubes, and counted by Cerenkov radiation.…”

Section: Methodsmentioning

confidence: 99%

“…In bovine heart, two alternatively spliced forms have been described (15,16), migrating with M r values of 58,000 and 54,000 on SDS-polyacrylamide gels (15). Peptide sequence analysis revealed that the M r 54,000 form lacks phosphorylation sites for PKA (Ser-466 and Ser-483) and protein kinase C (Ser-466 and Thr-475), which are encoded by exon 15 and thus present in the M r 58,000 form (15,17). The situation in bovine heart is further complicated by the fact that four different mRNAs have been characterized (18).…”

mentioning

confidence: 99%

Phosphorylation and Activation of Heart 6-Phosphofructo-2-kinase by Protein Kinase B and Other Protein Kinases of the Insulin Signaling Cascades

Deprez¹,

Vertommen²,

Alessi³

et al. 1997

Journal of Biological Chemistry

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385

271

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To understand the insulin-induced activation of 6-phosphofructo-2-kinase (PFK-2) of the bifunctional enzyme PFK-2/fructose-2,6-bisphosphatase in heart, the effect of phosphorylation by protein kinases of the insulin signaling pathways on PFK-2 activity was studied. Purified PFK-2/fructose-2,6-bisphosphatase from bovine heart is a mixture of two isoforms (M r 58,000 and 54,000 on SDS-polyacrylamide gels). The M r 54,000 protein is an alternatively spliced form, lacking phosphorylation sites for protein kinases. Recombinant enzymes corresponding to the M r 58,000 (BH1) and M r 54,000 (BH3) forms were expressed and used as substrates for phosphorylation. The recombinant BH1 isoform was phosphorylated by p70 ribosomal S6 kinase (p70 s6k ), mitogenactivated protein kinase-activated protein kinase-1, and protein kinase B (PKB), whereas the recombinant BH3 isoform was a poor substrate for these protein kinases. Treatment with all protein kinases activated PFK-2 in the recombinant BH1 preparation. Phosphorylation of the recombinant BH1 isoform correlated with PFK-2 activation and was reversed by treatment with protein phosphatase 2A. All the protein kinases phosphorylated Ser-466 and Ser-483 in the BH1 isoform, but to different extents: p70 s6k preferentially phosphorylated Ser-466, whereas mitogen-activated protein kinase-activated protein kinase-1 and PKB phosphorylated Ser-466 and Ser-483 to a similar extent. We propose that PKB is part of the insulin signaling cascade for PFK-2 activation in heart.

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Evidence for new phosphorylation sites for protein kinase C and cyclic AMP‐dependent protein kinase in bovine heart 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase

Cited by 26 publications

References 13 publications

Cloning and expression of novel isoforms of 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase from bovine heart

Cloning and expression of novel isoforms of 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase from bovine heart

Identification of Phosphorylation Sites in AMP-activated Protein Kinase (AMPK) for Upstream AMPK Kinases and Study of Their Roles by Site-directed Mutagenesis

Phosphorylation and Activation of Heart 6-Phosphofructo-2-kinase by Protein Kinase B and Other Protein Kinases of the Insulin Signaling Cascades

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