Tissue Transglutaminase Has Intrinsic Kinase Activity

Abstract

Tissue transglutaminase (TG2) is a ubiquitous enzyme that cross-links glutamine residues with lysine residues, resulting in protein polymerization, cross-linking of dissimilar proteins, and incorporation of diamines and polyamines into proteins. It has not previously been known to have kinase activity. Recently, insulin-like growth factor-binding protein-3 (IGFBP-3) has been reported to be phosphorylated by breast cancer cell membranes. We purified the IGFBP-3 kinase activity from solubilized T47D breast cancer cell membranes using gel filtration, ion-exchange chromatography, and IGFBP-3 affinity chromatography. The fractions containing kinase activity were further purified by high pressure liquid chromatography and analyzed by tandem mass spectroscopy. TG2 was detected in fractions containing kinase activity. Antisera to TG2 and protein A-Sepharose were used to immunoprecipitate TG2 from membrane fractions. The immunoprecipitates retained IG-FBP-3 kinase, whereas immunoprecipitation deleted kinase activity in the membrane supernatant. The inhibitors of TG2, cystamine and monodansyl cadaverine, abolished the ability of the T47D cell membrane preparation to phosphorylate IGFBP-3. Both TG2 purified from guinea pig liver and recombinant human TG2 expressed in insect cells were able to phosphorylate IGFBP-3. TG2 kinase activity was inhibited in a concentration-dependent fashion by calcium, which has previously been shown to be important for the cross-linking activity of TG2. These data provide compelling evidence that TG2 has intrinsic kinase activity, a function that has not previously been ascribed to TG2. Furthermore, we provide evidence that TG2 is a major component of the IGFBP-3 kinase activity present on breast cancer cell membranes.

Functional characterization of naturally occurring transglutaminase 2 mutants implicated in early-onset type 2 diabetes

“…We have recently found that IGFBP3 has a role in insulin secretion (Nguyen et al 2011). Moreover, we have shown that ATP and Ca 2C reciprocally regulate transamidation and kinase activities of TG2 (Mishra & Murphy 2004). ATP facilitates kinase activity and has an inhibitory effect on transamidation activity, whereas Ca 2C has an opposite effect (Mishra & Murphy 2004, Mishra et al 2007a.…”

“…5, 10 mM magnesium chloride, [g-32 P]ATP (25 mCi/ml)) for 30 min at 30 8C as described previously (Mishra & Murphy 2004, Mishra et al 2006. The reaction was stopped by the addition of SDS-PAGE sample buffer and the samples were boiled for 5 min.…”

“…Moreover, we have shown that ATP and Ca 2C reciprocally regulate transamidation and kinase activities of TG2 (Mishra & Murphy 2004). ATP facilitates kinase activity and has an inhibitory effect on transamidation activity, whereas Ca 2C has an opposite effect (Mishra & Murphy 2004, Mishra et al 2007a. Furthermore, TG2 binds to GTP and functions as a G-protein in cell signaling processes (Baek et al 2001), and the GTP-binding and transamidation functions of TG2 are inversely associated with each other (Murthy et al 2002, Begg et al 2006 ) binding loops are located distantly from each other in the primary structure of TG2, they are very close to each other in the secondary structure, as revealed by the crystal structure of TG2 (Murthy et al 2002, Begg et al 2006.…”

“…In addition, we have reported that TG2 has intrinsic kinase activity and phosphorylates a number of proteins including insulin-like growth factor binding protein-3 (IGFBP3; Mishra & Murphy 2004, Mishra et al 2006, 2007a. We have recently found that IGFBP3 has a role in insulin secretion (Nguyen et al 2011).…”

Tissue transglutaminase (TG2) protects cardiomyocytes against ischemia/reperfusion injury by regulating ATP synthesis

“…3 Recently a protein disulphide isomerase and a kinase activity of TG2 have also been described. 4,5 To assess the biological role, TG2 knockout mice have been generated. These mice show impaired glucose-stimulated insulin secretion and intolerance after glucose loading.…”