Phosphorylation of transglutaminase 2 by PKA at Ser216 creates 14-3-3 binding sites

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Abstract

Transglutaminase 2 (TG2) is a multifunctional ubiquitous enzyme which is present in various cellular compartments and is subject to phosphorylation by PKA. To better understand the relevance of PKA induced phosphorylation of TG2, we performed pull-down assays using phosphorylated biotinylated-TG2209–223 peptides spanning PKA induced phosphorylation sites as a bait. Subsequent analysis of pull-down protein by SDS–PAGE and LC/MS identified 14-3-3ε as the binding partner for TG2 which was further confirmed by immunoblotting with 14-3-3 specific antiserum. In contrast, non-phosphorylated and/or phosphorylation site substituted peptides fail to pull-down 14-3-3. Furthermore, we demonstrate that 14-3-3 co-immunoprecipitated with TG2 antiserum after activation of PKA from mouse embryonic fibroblasts (MEF)TG2+/+ cells but not from MEFTG2−/− cells. In summary, we provide convincing evidence that phosphorylation of TG2 by PKA creates binding site(s) for 14-3-3 both in vitro and in vivo.

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Materials and methods

Reagents. Recombinant PKA was obtained from New England Biolabs (Ipswich, MA, USA). Anti-TG2 antibody was purchased from Upstate Biotechnology (Lake Placid, NY, USA) and anti-14-3-3 antibody from Abcam Inc (Cambridge, MA, USA). Protein A–agarose was obtained from Pierce (Rockford, IL, USA). Dibutyryl-cAMP, streptavidin–HRP, streptavidin–agarose, and all other reagents unless otherwise stated were obtained from Sigma–Aldrich Canada (Oakville, Ont.). MCF-7 cells were obtained from American Type

Peptide synthesis

TG2209–223 and its mutated forms were chemically synthesized as 15 residue peptide amidated at C-terminus and biotinylated at N-terminus. The correct peptides were obtained in greater than 90% yield and were homogeneous after purification as confirmed by mass and analytical HPLC. The amino acid sequence of peptides is summarized in Table 1.

S125 and S216 in TG2 are phosphorylated by PKA

We have recently identified that TG2 is phosphorylated by PKA and using the TG2209–223 peptide we have shown that it is S216 which is the dominant

Acknowledgments

We thank Dr. G. Melino for providing us TG2−/− and TG2+/+ MEF cells. This research was supported by funds from the Canadian Institute of Health Research. L.J.M. is a recipient of the Henry G. Friesen Research Professorship in Endocrine and Metabolic Diseases.

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Abbreviations: PKA, protein kinase A; TG2, transglutaminase 2; MEF, mouse embryonic fibroblast; SDS–PAGE, sodium dodecyl sluphate–polyacrylamide gel electrophoresis; Stvn, Streptavidin; HRP, horseradish peroxidase; Biot, biotinylated; ECL, enhanced chemiluminescence.

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