Tissue Transglutaminase Has Intrinsic Kinase Activity

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 IGFBP-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. 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 IGFBP-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. IntroductionInsulin-like growth factor-binding protein-3 (IGFBP-3) 1The abbreviations used are: IGFBP-3, insulin-like growth factor-binding protein-3; MDC, monodansyl cadaverine. is the most abundant of the IGFBPs in the circulation. It is a multifunctional protein that not only transports insulin-like growth factors (IGF)-I and -II and modulates the actions of these growth factors but also has IGF-independent anti-proliferative and proapoptotic effects (1Jones J.I. Clemmons D.R. Endocr. Rev. 1995; 16: 3-34Google Scholar).IGFBP-3 can both enhance and inhibit the effects of IGF-I in vivo and in vitro depending upon experimental conditions (2DeMellow J.S.M. Baxter R.C. Biochem. Biophys. Res. Commun. 1988; 156: 199-204Google Scholar, 3Valentinis B. Bhala A. DeAngelis T. Baserga R. Cohen P. Mol. Endocrinol. 1996; 9: 361-367Google Scholar, 4Oh Y. Gucev Z. Ng L. Muller H.L. Rosenfeld R.G. Prog. Growth Factor Res. 1995; 6: 205-212Google Scholar, 5Lalou C. Lassarre C. Binoux M.A. Endocrinology. 1996; 137: 3206-3212Google Scholar, 6Hong J. Zhang G. Dong F. Rechler M.M. J. Biol. Chem. 2002; 277: 10489-10497Google Scholar). Enhancement of IGF-I action may result from enhanced delivery of IGF-I to its plasma membrane receptor, whereas inhibition may be a consequence of competition between IGFBP-3 and the type 1 IGF receptor for binding of IGF-I (1Jones J.I. Clemmons D.R. Endocr. Rev. 1995; 16: 3-34Google Scholar, 2DeMellow J.S.M. Baxter R.C. Biochem. Biophys. Res. Commun. 1988; 156: 199-204Google Scholar).In addition to these IGF-dependent effects, emerging evidence suggests that IGFBP-3 also functions directly to stimulate apoptosis and inhibit cellular proliferation of various cell lines, including human breast cancer cells (4Oh Y. Gucev Z. Ng L. Muller H.L. Rosenfeld R.G. Prog. Growth Factor Res. 1995; 6: 205-212Google Scholar). However, these IGF-independent effects are only apparent under conditions where the IGF-I-dependent effects are not observed, such as studies with mutant IGFBP-3 and IGFBP-3 fragments that have minimal affinity for IGF-I (5Lalou C. Lassarre C. Binoux M.A. Endocrinology. 1996; 137: 3206-3212Google Scholar, 6Hong J. Zhang G. Dong F. Rechler M.M. J. Biol. Chem. 2002; 277: 10489-10497Google Scholar) and with cell lines devoid of IGF-I receptors (3Valentinis B. Bhala A. DeAngelis T. Baserga R. Cohen P. Mol. Endocrinol. 1996; 9: 361-367Google Scholar).To further understand the mechanisms that allow for these opposing effects of IGFBP-3, we have investigated the interaction of IGFBP-3 with breast cancer cell membranes. In addition to proteolysis we have recently reported that IGFBP-3 is phosphorylated by breast cancer cells by a process that occurs on the cell membranes, does not require internalization, and is inhibited by IGF-I (7Mishra S. Murphy L.J. Endocrinology. 2003; 144: 4042-4050Google Scholar). Phosphorylation of IGFBP-3 by this membrane-associated kinase enhanced the binding affinity of IGFBP-3 for IGF-I (7Mishra S. Murphy L.J. Endocrinology. 2003; 144: 4042-4050Google Scholar). Thus, phosphorylation of IGFBP-3 at the membrane favors the interaction of IGF-I with IGFBP-3 rather than the IGF-I receptor. Furthermore, because formation of IGF-I·IGFBP-3 complexes inhibits binding of IGFBP-3 to the cell membrane, phosphorylation of IGFBP-3 may modulate its pro-apoptotic anti-proliferative effects. To further understand the role of this kinase in physiological regulation of IGFBP-3 action, we purified this kinase activity from T47D breast cancer cells. Here we report that this kinase activity is attributable to TG2.TG2 is a widely expressed enzyme that is involved in polymerization and aggregation of proteins via cross-linking glutamine residues. Although it contains a GTP binding domain and can hydrolyze both GTP and ATP (8Lai T.S. Slaughter T.F. Peoples K.A. Hettasch J.M. Greenberg C.S. J. Biol. Chem. 1998; 273: 1776-1781Google Scholar), it has not previously been reported to have kinase activity.EXPERIMENTAL PROCEDURESMaterials and Reagents—T47D and MCF-7 cells were obtained from the American Type Tissue Collection (Manassas, VA). Cell culture reagents were from Invitrogen. Glycosylated and non-glycosylated IGFBP-3 were obtained from Upstate Biotechnology Inc. (Lake Placid, NY). Recombinant TG2 was purchased from Roboscreen. Guinea pig liver TG, the inhibitors cystamine and monodansyl cadaverine (MDC), biotinylated phospho-specific monoclonal antibodies, streptavidinhorseradish peroxidase conjugate and all other reagents, unless otherwise stated, were obtained from Sigma-Aldrich.Biotinylation of IGFBP-3—Non-glycosylated Escherichia coli-derived IGFBP-3 was biotinylated using p-biotinoyl-aminocaproic acid-N-hydroxy-succinamide ester (Roche Applied Science) as previously described (7Mishra S. Murphy L.J. Endocrinology. 2003; 144: 4042-4050Google Scholar).Purification of IGFBP-3 Kinase Activity—Solubilized T47D cell membranes were prepared using a membrane preparation kit (Pierce, Rockford, IL) according to the manufacturer's instructions in the presence of protease inhibitors (0.1 mm phenylmethylsulfonyl fluoride, 10 mm aprotinin, and 10 μg/ml leupeptin). 3 ml of solubilized membranes was filtered through a 0.22-μm filter and loaded on a 16/60 Sephacryl S-100 gel filtration column that had been equilibrated with 20 mm Tris, 0.02% NaN3, pH 7.5. The eluate was monitored for absorbance at 280 nm through a Pharmacia UV-1 single path monitor. 1-ml fractions were collected at a flow rate of 1 ml/min and stored at –70 C. The activity was consistently found in fractions within molecular mass range of 65–85 kDa. A 20-μl aliquot of each fraction was assayed for IGFBP-3 kinase activities. Active fractions were pooled and concentrated with an Amicon Centricon 30 filter. A buffer-exchanged sample was passed through a High Q anion exchange column (Bio-Rad) that had been equilibrated with 50 mm Tris, pH 8.0, containing 0.05 m NaCl, 0.02% NaN3. Separation was performed in a linear gradient from 0.05–0.5 m NaCl over 50 min at flow rate of 1 ml/min, and 1-ml fractions were collected. Fractions containing kinase activity were concentrated using an Amicon Centricon filter and desalted. The buffer was exchanged using Micro Bio-Spin chromatography columns (Bio-Rad) and loaded onto an IGFBP-3-Sepharose 4B affinity column (2-ml bed volume). Bound proteins were first eluted with 0.05 m sodium phosphate containing 0.15 m NaCl, pH 7.2, followed by 0.1 m acetate buffer containing 0.5 m NaCl, pH 4.0. Eluted fractions were desalted, concentrated, and used for IGFBP-3 kinase assay. A fraction with IGFBP-3 kinase activity was processed for liquid chromatography mass spectroscopy.For liquid chromatography mass spectroscopy analysis, 100 μl of desalted affinity fraction was digested with sequencing grade trypsin. The peptide mixture was lyophilized and resuspended in 10 μl of 0.05% trifluoroacetic acid and used for μHPLC-matrix-assisted laser desorption ionization quadrupole time-of-flight analysis. Chromatographic separation was performed using an Agilent 1100 Series system. Samples (5 μl) were injected into a 150 μm × 150 mm column (5μ, Vydac 218 TP C18) and eluted with trifluoroacetic in of the were analyzed by mass in a quadrupole time-of-flight mass In this are by of the using from a the mass is within a in time-of-flight of the was by data the peptide using the of were with and with in and stored at IGFBP-3 was in for on the of IGFBP-3 was were on and a phosphorylation mixture containing 20 mm pH 10 mm was was by the addition of a membrane fraction and to for 30 min at 30 was by the addition of sample for and analyzed on were and processed for In of guinea pig liver TG2 human TG2 expressed using the in insect cells and purified by chromatography was used to phosphorylate IGFBP-3 in the presence of To the of IGFBP-3 the was in a of μl containing 10 of and of were at various with sample and for proteins was on and were with to phosphorylated IGFBP-3 were and were by linear analysis. the phosphate was into IGFBP-3 at various in the presence of of transglutaminase for In where cell were used to phosphorylate IGFBP-3, cells were in culture to and with to and Phosphorylation was performed in 100 μl of mixture as containing 1 of IGFBP-3 for 10 min at the of the mixture was and the was by the addition of sample buffer and analyzed on In cell were with TG2 for 30 min to the phosphorylation of Phosphorylation in phosphorylation in IGFBP-3 were by the under conditions and by the using of and phosphorylation within the IGFBP-3 IGFBP-3 and IGFBP-3 and were phosphorylated using recombinant TG2. proteins and were on to membranes, and with monoclonal at membranes were with streptavidinhorseradish peroxidase conjugate for 1 at and detected using the μl of was to μl of solubilized membranes from T47D MCF-7 cells and for 1 at 20 μl of was and further on a at the of the was in The was and the was resuspended in 50 μl of kinase 10 μl of the resuspended sample was used for phosphorylation of of IGFBP-3. The were analyzed by and with TG2 column fractions that had IGFBP-3 kinase activity were analyzed on gel and to membranes. were in with TG2 to (5 min in mm Tris, 150 mm NaCl, 0.05% pH and with for 1 at were in and analyzed with of IGFBP-3 Kinase from T47D Cell IGFBP-3 kinase activity was purified from solubilized T47D cell membranes using biotinylated IGFBP-3 as a A was used gel and IGFBP-3 affinity chromatography Fractions eluted from the IGFBP-3 affinity column under conditions that IGFBP-3 kinase activity were further analyzed by high pressure liquid chromatography and tandem mass spectroscopy. the a of proteins were that had TG2 had the and was over the TG2 with from various of the mass spectroscopy was used to the of various peptide fragments peptide fragments were by mass spectroscopy in IGFBP-3 affinity column fraction containing kinase as where is the that the is a A domain containing receptor as where is the that the is a A in a peptide by tandem mass spectroscopy in IGFBP-3 affinity column fraction containing kinase in a Fractions containing IGFBP-3 kinase activity from the various were analyzed by using TG2 TG2 was present in all was also in T47D cell membrane the of the TG2 and inhibitors on phosphorylation of IGFBP-3 by cell We have previously shown that cells were able to phosphorylate IGFBP-3 on (7Mishra S. Murphy L.J. Endocrinology. 2003; 144: 4042-4050Google Scholar). T47D cell were for 30 min in the presence of various of an of TG2. The cell were with IGFBP-3 in the presence of for 10 min at of the the IGFBP-3 was analyzed by and was with as as 20 and inhibition was apparent with 50 μm of TG2 has been found in with a J. Scholar), and was present in the cell membrane we also investigated the of the The had on IGFBP-3 were membrane from both T47D and MCF-7 cells. reported both T47D and MCF-7 cells were able to phosphorylate IGFBP-3. process was inhibited by cystamine and in both cells lines the in which was had The had on IGFBP-3 phosphorylation of TG2 and inhibitors on phosphorylation of IGFBP-3 by breast cancer cell cell membranes and various of cystamine the were with T47D cell to the ability of the cell to phosphorylate IGFBP-3. cystamine monodansyl cadaverine the was to MCF-7 and T47D cell membranes, and the ability of these membranes to phosphorylate IGFBP-3 was the ability of cystamine and 20 and the and to inhibit phosphorylation of IGFBP-3 by T47D cell membranes was the of TG2 and on IGFBP-3 kinase activity present in cell membrane TG2, but not was able to IGFBP-3 kinase activity from membrane the immunoprecipitates were analyzed for kinase activity, the obtained with TG2 but not obtained with had kinase activity. Furthermore, the IGFBP-3 kinase activity present in the immunoprecipitates was inhibited by cystamine but not by the and immunoprecipitation of IGFBP-3 kinase activity from T47D solubilized membranes. solubilized T47D cell membranes were with to TG2 the immunoprecipitates were using protein the were for the ability to phosphorylate IGFBP-3. TG2 and were from solubilized MCF-7 T47D cell membranes and analyzed on To the presence of these proteins in the the membrane was with the presence of IGFBP-3 kinase activity in the immunoprecipitates was and ability of cystamine and the (5 to inhibit this kinase activity was guinea pig liver TG2 and human recombinant TG2 both were able to phosphorylate IGFBP-3 process was inhibited by cystamine and The binding protein was also phosphorylated by TG2, whereas was not phosphorylated by TG2 with a report Clemmons D.R. J. Biol. Chem. Scholar), an in the molecular mass of was observed, that TG2 can not reported for TG2 J. Cell Scholar), was not phosphorylated by this enzyme under the conditions we used to phosphorylate IGFBP-3 guinea pig liver TG2 and recombinant human TG2 have IGFBP-3 kinase activity. the of cystamine 20 and on the IGFBP-3 kinase activity of guinea pig and recombinant TG2 was and with only the ability of recombinant human TG2 to phosphorylate and fragments was the of on human recombinant TG2 kinase activity and cross-linking activity was the of on TG2 kinase activity is shown In the IGFBP-3 in the of TG2 and was In the the has been with The the of and is for the cross-linking activity of TG2 Biochem. Biophys. Res. Commun. 2002; Scholar), we the of of on the kinase activity of TG2. the was we a in the ability of TG2 to phosphorylate IGFBP-3 was an in polymerization of IGFBP-3 IGFBP-3 was only phosphorylated with IGFBP-3 of phosphorylation of IGFBP-3 by TG2 was conditions of was of IGFBP-3, the presence of at TG2 phosphorylation in IGFBP-3. The and for the TG2 phosphorylation was μm and of IGFBP-3, and of phosphorylation of IGFBP-3 by TG2. TG2 phosphorylation of IGFBP-3 was as described under were at various the phosphate was into IGFBP-3 at various in the presence of of transglutaminase for of IGFBP-3 with a with a with and with not under a of and residues were and in the residues at only phosphorylation were and were to residues and residues and the of phosphorylation These all phosphorylation with the of in peptide and and Both were phosphorylated by TG2, and of that peptide 1 for of the phosphorylation of IGFBP-3, whereas peptide for Both were by an to residues to was also by an is and in peptide 1 is not most by is of IGFBP-3 and IGFBP-3 by TG2. of IGFBP-3 IGFBP-3 1 and were phosphorylated with TG2 and analyzed on proteins were to membranes and with IGFBP-3 was as a In was and phosphorylation was detected by of phosphorylation in human from The the is to the the an phosphorylation in within 1 from The the is to the the an phosphorylation within 1 in a of purified fractions from breast cancer cells containing IGFBP-3 kinase activity a of proteins. these only was previously known to have kinase activity, and we that this was for phosphorylation of IGFBP-3. However, a of kinase activity had on the IGFBP-3 kinase activity of breast cancer cell purified membrane Furthermore, immunoprecipitation of from membrane preparation not the IGFBP-3 kinase activity, whereas this activity be by immunoprecipitation with TG2 and by TG2 These with the that both guinea pig liver TG2 and recombinant human TG2 phosphorylate IGFBP-3, evidence that TG2 can function as an in breast cancer cells. Furthermore, it to for all the IGFBP-3 kinase activity present on the membrane of these cells because activity was apparent immunoprecipitation of TG2 from breast cancer membrane We have previously shown that IGFBP-3 can also be phosphorylated by an present on cells (7Mishra S. Murphy L.J. Endocrinology. 2003; 144: 4042-4050Google Scholar) and human cells. and L. J. The is because cells are known to high of TG2 on plasma membranes L. Biochem. 2002; of IGFBP-3 by TG2 was at the residues in the domain of the domain with which is also phosphorylated by TG2. In the domain of is from IGFBP-3, and this for the of TG2 to phosphorylate and J. Biochem. Scholar) report that to at residues and in an of phosphorylated IGFBP-3 by cells. However, the kinase was not and it is not such have on of other residues for It is that and are the of TG2 phosphorylation in peptide However, peptide 1 only for of the phosphorylation of IGFBP-3 by TG2. residues further in peptide were also phosphorylated by TG2. In addition to we provide evidence that TG2 also in phosphorylation at a most is a ubiquitous enzyme that has been in a of It is important in protein and It functions as a that cross-links glutamine residues with lysine residues in the proteins, resulting in polymerization, with lysine residues in other proteins, resulting in protein cross-linking L. Biochem. 2002; Scholar). In addition to diamines and polyamines to proteins, it can also glutamine residues to which a and the of the it has been reported to also function as a protein G. Y. T. S. Y. Y. Biochem. J. 2003; Scholar). However, this other functions described for TG2, was not and was not inhibited by TG2 has also been reported to function as membrane receptor T. A. Scholar) and has been shown to have a role in from receptors such as the receptor S. F. S. J. Biol. Chem. 1996; Scholar). Here we report that TG2 has kinase activity. Although we have as only IGFBP-3 and the protein as for this kinase activity, it is to phosphorylate a of other the kinase activity of TG2 was inhibited by with Biochem. Biophys. Res. Commun. 2002; Scholar), the enhanced the cross-linking activity of TG2. In the of TG2 activity IGFBP-3, to as a kinase activity and cross-linking activity. It is not from studies high inhibit TG2 kinase activity directly rather enhance TG2 cross-linking of activity and phosphorylation by the of phosphorylation in IGFBP-3. The because IGFBP-3 is only phosphorylated with IGFBP-3 has been in a of where phosphorylation is These of and protein kinase J. S. J. Biol. Chem. 2003; Scholar), of protein J. J. Biol. Chem. 2003; Scholar), and of T. A. Scholar). It is the kinase activity of TG2 is important in these most cell TG2 is in the and the L. Biochem. 2002; Scholar), but it is also to the cell membrane A. C. J. Biol. Chem. Scholar). It can be from various cell under such as and cell J.M. Tissue in Cell in Cell in and Scholar). In the it to be important in the of the process and may function to cells for by J.M. Tissue in Cell in Cell in and Scholar). However, TG2 is in apoptosis in Cell 6: Scholar) and apoptosis D.R. Prog. Biol. Res. IGFBP-3 has been shown to be pro-apoptotic in a of cell lines (4Oh Y. Gucev Z. Ng L. Muller H.L. Rosenfeld R.G. Prog. Growth Factor Res. 1995; 6: 205-212Google Scholar, 6Hong J. Zhang G. Dong F. Rechler M.M. J. Biol. Chem. 2002; 277: 10489-10497Google Scholar, L. C. L. F. Rosenfeld R.G. A. Endocrinology. 2003; 144: Scholar). process is to be an IGF-independent of IGFBP-3 by binding of IGFBP-3 to a receptor Y. Muller H.L. Rosenfeld R.G. J. Biol. Chem. Scholar). The presence of IGF-I inhibits the interaction of IGFBP-3 with binding present on breast cancer cells Y. Rosenfeld R.G. Y. Endocrinology. Scholar) and inhibit the pro-apoptotic IGF-independent effects of IGFBP-3. We have previously shown that phosphorylation of IGFBP-3 by TG2 the affinity of this binding protein for Thus, phosphorylation of IGFBP-3 by TG2 to the pro-apoptotic effects of IGFBP-3 and the of IGF-I by of complexes and the interaction of IGF-I and IGFBP-3 with membrane binding we have a kinase function for TG2. We provide compelling evidence that TG2 is the major IGFBP-3 kinase present on breast cancer cell membranes. The that TG2 has kinase activity as a to the role of TG2 kinase activity in other where TG2 activity be IntroductionInsulin-like growth factor-binding protein-3 (IGFBP-3) 1The abbreviations used are: IGFBP-3, insulin-like growth factor-binding protein-3; MDC, monodansyl cadaverine. is the most abundant of the IGFBPs in the circulation. It is a multifunctional protein that not only transports insulin-like growth factors (IGF)-I and -II and modulates the actions of these growth factors but also has IGF-independent anti-proliferative and proapoptotic effects (1Jones J.I. Clemmons D.R. Endocr. Rev. 1995; 16: 3-34Google Scholar).IGFBP-3 can both enhance and inhibit the effects of IGF-I in vivo and in vitro depending upon experimental conditions (2DeMellow J.S.M. Baxter R.C. Biochem. Biophys. Res. Commun. 1988; 156: 199-204Google Scholar, 3Valentinis B. Bhala A. DeAngelis T. Baserga R. Cohen P. Mol. Endocrinol. 1996; 9: 361-367Google Scholar, 4Oh Y. Gucev Z. Ng L. Muller H.L. Rosenfeld R.G. Prog. Growth Factor Res. 1995; 6: 205-212Google Scholar, 5Lalou C. Lassarre C. Binoux M.A. Endocrinology. 1996; 137: 3206-3212Google Scholar, 6Hong J. Zhang G. Dong F. Rechler M.M. J. Biol. Chem. 2002; 277: 10489-10497Google Scholar). Enhancement of IGF-I action may result from enhanced delivery of IGF-I to its plasma membrane receptor, whereas inhibition may be a consequence of competition between IGFBP-3 and the type 1 IGF receptor for binding of IGF-I (1Jones J.I. Clemmons D.R. Endocr. Rev. 1995; 16: 3-34Google Scholar, 2DeMellow J.S.M. Baxter R.C. Biochem. Biophys. Res. Commun. 1988; 156: 199-204Google Scholar).In addition to these IGF-dependent effects, emerging evidence suggests that IGFBP-3 also functions directly to stimulate apoptosis and inhibit cellular proliferation of various cell lines, including human breast cancer cells (4Oh Y. Gucev Z. Ng L. Muller H.L. Rosenfeld R.G. Prog. Growth Factor Res. 1995; 6: 205-212Google Scholar). However, these IGF-independent effects are only apparent under conditions where the IGF-I-dependent effects are not observed, such as studies with mutant IGFBP-3 and IGFBP-3 fragments that have minimal affinity for IGF-I (5Lalou C. Lassarre C. Binoux M.A. Endocrinology. 1996; 137: 3206-3212Google Scholar, 6Hong J. Zhang G. Dong F. Rechler M.M. J. Biol. Chem. 2002; 277: 10489-10497Google Scholar) and with cell lines devoid of IGF-I receptors (3Valentinis B. Bhala A. DeAngelis T. Baserga R. Cohen P. Mol. Endocrinol. 1996; 9: 361-367Google Scholar).To further understand the mechanisms that allow for these opposing effects of IGFBP-3, we have investigated the interaction of IGFBP-3 with breast cancer cell membranes. In addition to proteolysis we have recently reported that IGFBP-3 is phosphorylated by breast cancer cells by a process that occurs on the cell membranes, does not require internalization, and is inhibited by IGF-I (7Mishra S. Murphy L.J. Endocrinology. 2003; 144: 4042-4050Google Scholar). Phosphorylation of IGFBP-3 by this membrane-associated kinase enhanced the binding affinity of IGFBP-3 for IGF-I (7Mishra S. Murphy L.J. Endocrinology. 2003; 144: 4042-4050Google Scholar). Thus, phosphorylation of IGFBP-3 at the membrane favors the interaction of IGF-I with IGFBP-3 rather than the IGF-I receptor. Furthermore, because formation of IGF-I·IGFBP-3 complexes inhibits binding of IGFBP-3 to the cell membrane, phosphorylation of IGFBP-3 may modulate its pro-apoptotic anti-proliferative effects. To further understand the role of this kinase in physiological regulation of IGFBP-3 action, we purified this kinase activity from T47D breast cancer cells. Here we report that this kinase activity is attributable to TG2.TG2 is a widely expressed enzyme that is involved in polymerization and aggregation of proteins via cross-linking glutamine residues. Although it contains a GTP binding domain and can hydrolyze both GTP and ATP (8Lai T.S. Slaughter T.F. Peoples K.A. Hettasch J.M. Greenberg C.S. J. Biol. Chem. 1998; 273: 1776-1781Google Scholar), it has not previously been reported to have kinase activity.