Biochemical and Biophysical Research Communications
Phosphorylation of transglutaminase 2 by PKA at Ser216 creates 14-3-3 binding sites☆
Section snippets
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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High-throughput investigation of transglutaminase 2 kinase regulation using a novel cysteine-modified peptide array
2018, Analytical BiochemistryActivation of transglutaminase 2 by nerve growth factor in differentiating neuroblastoma cells: A role in cell survival and neurite outgrowth
2018, European Journal of PharmacologyCitation Excerpt :At present it is not known if ERK1/2, PKB and PKC directly phosphorylate TG2, leading to direct enhancement of enzymic activity or whether TG2 phoshorylation promotes its association with interacting proteins. For example, TG2 phosphorylation by PKA promotes its interaction with the scaffolding protein 14-3-3 which leads to the attenuation of TG2 kinase activity (Mishra and Murphy, 2006). Alternatively, these kinases may phosphorylate downstream targets that subsequently interact with TG2, resulting in enhanced activity.
β<inf>2</inf>-adrenoceptor-induced modulation of transglutaminase 2 transamidase activity in cardiomyoblasts
2017, European Journal of PharmacologyCitation Excerpt :At present it is not known if formoterol triggers TG2 phosphorylation at Ser215 and Ser216. However, previous studies have revealed that PKA-mediated phosphorylation of TG2 at these sites has several potential consequences, including promotion of protein-protein interactions, enhancement of TG2 kinase activity and inhibition of transamidating activity (Mishra and Murphy, 2006; Mishra et al., 2007). In this study β2-adrenoceptor-induced PKA activation promoted TG2 transamidating activity.
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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.