The Experts below are selected from a list of 228 Experts worldwide ranked by ideXlab platform
George M. Carman - One of the best experts on this subject based on the ideXlab platform.
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Phosphatidylinositol 4-kinases in Saccharomyces cerevisiae
Advances in Lipobiology, 1996Co-Authors: George M. Carman, Rosa J. Buxeda, Joseph T. NickelsAbstract:Abstract Phosphatidylinositol 4-kinase from the yeast Saccharomyces cerevisiae catalyzes the formation of phosphatidylinositol 4-phosphate and ADP from phosphatidylinositol and ATP. Phosphatidylinositol 4-kinase catalyzes the first phosphorylation reaction in the reaction sequence phosphatidylinositol → phosphatidylinositol 4-phosphate → phosphatidylinositol 4,5-bisphosphate. This phosphorylation sequence in S. cerevisiae is regulated by glucose and sterol. Phosphatidylinositol 4,5-bisphosphate appears to play an essential role in cell proliferation of S. cerevisiae . Since phosphatidylinositol 4-kinase catalyzes the first step in the phosphorylation sequence of phosphatidylinositol, the enzyme should play a major role in phosphoinositide synthesis and cell growth in S. cerevisiae . Two membrane-associated (45 kDa and 55 kDa) forms and one cytosolic-associated (125 kDa) form of phosphatidylinositol 4-kinase have been purified and characterized from S. cerevisiae . The membrane-associated phosphatidylinositol 4-kinases differ with respect to their physiochemical, enzymological, and kinetic properties. The binding and catalytic steps of the reactions catalyzed by the membrane-associated phosphatidylinositol 4-kinases toward phosphatidylinositol has been been defined through meaningful kinetic analyses using Triton X-100/PI-mixed micelles. Detailed kinetic analyses of the inhibition of membrane-associated phosphatidylinositol 4-kinases by nucleotides has led to insights on the regulation of phosphoinositide synthesis in response to glucose.
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Purification, characterization, and kinetic analysis of a 55-kDa form of phosphatidylinositol 4-kinase from Saccharomyces cerevisiae.
The Journal of biological chemistry, 1992Co-Authors: Joseph T. Nickels, Rosa J. Buxeda, George M. CarmanAbstract:Abstract A 55-kDa form of membrane-associated phosphatidylinositol 4-kinase (ATP:phosphatidylinositol 4-phosphotransferase, EC 2.7.1.67) was purified 10,166-fold from Saccharomyces cerevisiae. The purification procedure included solubilization of microsome membranes with 1% Triton X-100 followed by chromatography with DE52, hydroxylapatite I, Q-Sepharose, Mono Q, and hydroxylapatite II. The procedure resulted in a nearly homogeneous 55-kDa phosphatidylinositol 4-kinase preparation. The 55-kDa phosphatidylinositol 4-kinase and the previously purified 45-kDa phosphatidylinositol 4-kinase differed with respect to their amino acid composition, isoelectric points, and peptide maps. Furthermore, the two forms of phosphatidylinositol 4-kinase did not show an immunological relationship. Maximum 55-kDa phosphatidylinositol 4-kinase activity was dependent on magnesium (10 mM) or manganese (0.5 mM) ions and Triton X-100 at the pH optimum of 7.0. The activation energy for the reaction was 12 kcal/mol, and the enzyme was labile above 30 degrees C. The enzyme was inhibited by thioreactive agents, MgADP, and calcium ions. A detailed kinetic analysis of the purified enzyme was performed using Triton X-100/phosphatidylinositol-mixed micelles. 55-kDa phosphatidylinositol 4-kinase activity followed saturation kinetics with respect to the bulk and surface concentrations of phosphatidylinositol and followed surface dilution kinetics. The interfacial Michaelis constant (Km) and the dissociation constant (Ks) for phosphatidylinositol in the Triton X-100 micelle surface were 1.3 mol % and 0.035 mM, respectively. The Km for MgATP was 0.36 mM. 55-kDa phosphatidylinositol 4-kinase catalyzed a sequential reaction mechanism as indicated by the results of kinetic and isotopic exchange reactions. The enzyme bound to phosphatidylinositol before ATP and released phosphatidylinositol 4-phosphate before ADP. The enzymological and kinetic properties of the 55-kDa phosphatidylinositol 4-kinase differed significantly from those of the 45-kDa phosphatidylinositol 4-kinase. This may suggest that the two forms of phosphatidylinositol 4-kinase from S. cerevisiae are regulated differentially in vivo.
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Phosphatidylinositol 4-kinase from yeast.
Methods in enzymology, 1992Co-Authors: George M. Carman, Charles J. Belunis, Joseph T. NickelsAbstract:Publisher Summary Phosphatidylinositol 4-kinase (1-phosphatidylinositol kinase) catalyzes the reaction of phosphatidylinositol with ATP to form phosphatidylinositol 4-phosphate. Phosphatidylinositol 4-kinase (ATP:1-phosphatidyl-lD-myo-inositol 4-phosphotransferase) is the first enzyme in the phosphorylation sequence of phosphatidylinositol leading to the formation of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate in Saccharomyces cerevisiae (S. cerevisiae). The synthesis and turnover of the polyphosphoinositides in S. cerevisiae and in higher eukaryotes play an important role in cell growth. Phosphatidylinositol 4-kinase is associated with the microsomal, plasma membrane, and cytosolic fractions of S. cerevisiae. Phosphatidylinositol 4-kinase has been purified to near homogeneity from the microsomal fraction of S. cerevisiae by standard protein purification procedures. The chapter describes the purification and properties of the enzyme. The properties of phosphatidylinositol 4-kinase are also discussed. Pure phosphatidylinositol 4-kinase can be used to synthesize radiolabeled phosphatidylinositol 4-phosphate.
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Phosphatidylinositol synthase from yeast.
Methods in enzymology, 1992Co-Authors: George M. Carman, Anthony S. FischlAbstract:Publisher Summary Phosphatidylinositol synthase catalyzes the incorporation of inositol into phosphatidylinositol. The enzyme and its product phosphatidylinositol are essential to the growth of the yeast Saccharomyces cerevisiae ( S. cerevisiae ). Phosphatidylinositol synthase activity is associated with the mitochondrial, microsomal, and plasma membrane fractions of S. cerevisiae . Microsome-associated phosphatidylinositol synthase has been purified to near homogeneity, primarily by CDPdiacylglycerol-sepharose affinity chromatography. This chapter describes the purification, reconstitution, and properties of the enzyme. CDPdiacylglycerol is prepared from phosphatidic acid and CMPmorpholidate by the method of Agranoff and Suomi with the modifications of Carman and Fischl. Phosphatidic acid is prepared from soybean lecithin by reaction with cabbage phospholipase D. The chapter discusses the preparation of CDPdiacylglycerol-sepharose. Electroblotting of phosphatidylinositol synthase activity is described. Pure phosphatidylinositol synthase has been reconstituted into unilamellar phospholipid vesicles containing its substrate CDPdiacylglycerol. Pure phosphatidylinositol synthase can be used to synthesize radiolabeled phosphatidylinositol from CDPdiacylglycerol and labeled inositol.
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Phosphatidylinositol 4-kinase from Saccharomyces cerevisiae. Kinetic analysis using Triton X-100/phosphatidylinositol-mixed micelles.
The Journal of biological chemistry, 1991Co-Authors: Rosa J. Buxeda, Joseph T. Nickels, C J Belunis, George M. CarmanAbstract:Abstract Phosphatidylinositol 4-kinase (ATP:phosphatidylinositol 4-phosphotransferase, EC 2.7.1.67) was purified from Saccharomyces cerevisiae by an improved procedure over that previously reported (Belunis, C.J., Bae-Lee, M., Kelley, M.J., and Carman, G.M. (1988) J. Biol. Chem. 263, 18897-18903) for the enzyme. The molecular mass of the enzyme was 45 kDa. The 35-kDa protein previously identified as PI 4-kinase was a proteolysis product of the 45-kDa protein. A detailed kinetic analysis of the purified enzyme was performed with Triton X-100/phosphatidylinositol-mixed micelles according to the "surface dilution" (Deems, R.A., Eaton, B.R., and Dennis, E.A. (1975) J. Biol. Chem. 250, 9013-9020) and "dual phospholipid" (Hendrickson, H.S., and Dennis, E.A. (1984) J. Biol. Chem. 259, 5734-5739) kinetic models. Phosphatidylinositol 4-kinase activity followed saturation kinetics with respect to the bulk and surface concentrations of phosphatidylinositol at concentrations of phosphatidylinositol below 0.1 mM. Above 0.1 mM activity was only dependent on the surface concentration of phosphatidylinositol. The enzyme more closely followed the dual phospholipid model where the enzyme associated with Triton X-100 micelles when phosphatidylinositol was present. The interfacial Michaelis constant (KmB) for phosphatidylinositol was 0.0036 mol fraction and the dissociation constant (KsA) for phosphatidylinositol in the micelle surface was 0.26 mM. The results of glycerol gradient centrifugation studies showed that the enzyme was physically associated with Triton X-100/phosphatidylinositol micelles.
P. Leaky - One of the best experts on this subject based on the ideXlab platform.
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Effects of Ca^2+, Mg^2+, and depolarizing agents, on the^32Pi-labeling and degradation of Phosphatidylinositols in rat brain synaptosomes
Neurochemical Research, 1993Co-Authors: Guido V. Marinetti, T. W. Morris, P. LeakyAbstract:In isolated synaptosomes from rat brain, 100 μM antimycin A and 10 μM oxamic acid inhibit the^32Pi-labeling of phosphatidylinositol-4,5-bisphosphate (PIP_2) and phosphatidylinositol-4-phosphate (PIP) by 90% and 95–99% respectively. 10 mM sodium fluoride inhibits the labeling by 50–60% and 10 mM A23187 inhibits the labeling by 63–70%. Phospholipase A_2 inhibits the labeling of PIP_2 and PIP by 93–94% and stimulates their degradation by 84–92%. Depolarization of synaptosomes with 75 mM K^+ or 100 μM veratrine decreases the labeling of PIP_2 and PIP by 66–74%. The decreased labeling results in large part from the Ca^2+-dependent degradation of^32P-labeled PIP_2 and PIP as shown by pulse-chase experiments in which PIP_2 and PIP were prelabeled with^32Pi. Depolarization of synaptosomes results in the stimulation of^45Ca^2+ uptake with the concomitant hydrolysis of PIP and PIP_2. Addition of 1 mM Ca^2+ accounts for 25% of the enhanced degradation whereas depolarization with 75 mM K^+ accounts for 75% of the enhanced degradation of PIP_2 and PIP. Depolarization with 100 mM veratrine results in a 223% increase in inositol trisphosphate as evidenced by stimulation of^45Ca^2+ uptake. EGTA (10mM) and Mg^2+ (5–10 mM) inhibit the degradation of PIP and PIP_2 and counteract the action of 1 mM Ca^2+. Our data demonstrate that^45Ca^2+, Mg^2+, and membrane depolarization play an important role in the turnover of membrane Phosphatidylinositols.
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effects of ca2 mg2 and depolarizing agents on the32pi labeling and degradation of Phosphatidylinositols in rat brain synaptosomes
Neurochemical Research, 1993Co-Authors: Guido V. Marinetti, T. W. Morris, P. LeakyAbstract:In isolated synaptosomes from rat brain, 100 μM antimycin A and 10 μM oxamic acid inhibit the32Pi-labeling of phosphatidylinositol-4,5-bisphosphate (PIP2) and phosphatidylinositol-4-phosphate (PIP) by 90% and 95–99% respectively. 10 mM sodium fluoride inhibits the labeling by 50–60% and 10 mM A23187 inhibits the labeling by 63–70%. Phospholipase A2 inhibits the labeling of PIP2 and PIP by 93–94% and stimulates their degradation by 84–92%. Depolarization of synaptosomes with 75 mM K+ or 100 μM veratrine decreases the labeling of PIP2 and PIP by 66–74%. The decreased labeling results in large part from the Ca2+-dependent degradation of32P-labeled PIP2 and PIP as shown by pulse-chase experiments in which PIP2 and PIP were prelabeled with32Pi. Depolarization of synaptosomes results in the stimulation of45Ca2+ uptake with the concomitant hydrolysis of PIP and PIP2. Addition of 1 mM Ca2+ accounts for 25% of the enhanced degradation whereas depolarization with 75 mM K+ accounts for 75% of the enhanced degradation of PIP2 and PIP. Depolarization with 100 mM veratrine results in a 223% increase in inositol trisphosphate as evidenced by stimulation of45Ca2+ uptake. EGTA (10mM) and Mg2+ (5–10 mM) inhibit the degradation of PIP and PIP2 and counteract the action of 1 mM Ca2+. Our data demonstrate that45Ca2+, Mg2+, and membrane depolarization play an important role in the turnover of membrane Phosphatidylinositols.
Joseph T. Nickels - One of the best experts on this subject based on the ideXlab platform.
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Phosphatidylinositol 4-kinases in Saccharomyces cerevisiae
Advances in Lipobiology, 1996Co-Authors: George M. Carman, Rosa J. Buxeda, Joseph T. NickelsAbstract:Abstract Phosphatidylinositol 4-kinase from the yeast Saccharomyces cerevisiae catalyzes the formation of phosphatidylinositol 4-phosphate and ADP from phosphatidylinositol and ATP. Phosphatidylinositol 4-kinase catalyzes the first phosphorylation reaction in the reaction sequence phosphatidylinositol → phosphatidylinositol 4-phosphate → phosphatidylinositol 4,5-bisphosphate. This phosphorylation sequence in S. cerevisiae is regulated by glucose and sterol. Phosphatidylinositol 4,5-bisphosphate appears to play an essential role in cell proliferation of S. cerevisiae . Since phosphatidylinositol 4-kinase catalyzes the first step in the phosphorylation sequence of phosphatidylinositol, the enzyme should play a major role in phosphoinositide synthesis and cell growth in S. cerevisiae . Two membrane-associated (45 kDa and 55 kDa) forms and one cytosolic-associated (125 kDa) form of phosphatidylinositol 4-kinase have been purified and characterized from S. cerevisiae . The membrane-associated phosphatidylinositol 4-kinases differ with respect to their physiochemical, enzymological, and kinetic properties. The binding and catalytic steps of the reactions catalyzed by the membrane-associated phosphatidylinositol 4-kinases toward phosphatidylinositol has been been defined through meaningful kinetic analyses using Triton X-100/PI-mixed micelles. Detailed kinetic analyses of the inhibition of membrane-associated phosphatidylinositol 4-kinases by nucleotides has led to insights on the regulation of phosphoinositide synthesis in response to glucose.
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Purification, characterization, and kinetic analysis of a 55-kDa form of phosphatidylinositol 4-kinase from Saccharomyces cerevisiae.
The Journal of biological chemistry, 1992Co-Authors: Joseph T. Nickels, Rosa J. Buxeda, George M. CarmanAbstract:Abstract A 55-kDa form of membrane-associated phosphatidylinositol 4-kinase (ATP:phosphatidylinositol 4-phosphotransferase, EC 2.7.1.67) was purified 10,166-fold from Saccharomyces cerevisiae. The purification procedure included solubilization of microsome membranes with 1% Triton X-100 followed by chromatography with DE52, hydroxylapatite I, Q-Sepharose, Mono Q, and hydroxylapatite II. The procedure resulted in a nearly homogeneous 55-kDa phosphatidylinositol 4-kinase preparation. The 55-kDa phosphatidylinositol 4-kinase and the previously purified 45-kDa phosphatidylinositol 4-kinase differed with respect to their amino acid composition, isoelectric points, and peptide maps. Furthermore, the two forms of phosphatidylinositol 4-kinase did not show an immunological relationship. Maximum 55-kDa phosphatidylinositol 4-kinase activity was dependent on magnesium (10 mM) or manganese (0.5 mM) ions and Triton X-100 at the pH optimum of 7.0. The activation energy for the reaction was 12 kcal/mol, and the enzyme was labile above 30 degrees C. The enzyme was inhibited by thioreactive agents, MgADP, and calcium ions. A detailed kinetic analysis of the purified enzyme was performed using Triton X-100/phosphatidylinositol-mixed micelles. 55-kDa phosphatidylinositol 4-kinase activity followed saturation kinetics with respect to the bulk and surface concentrations of phosphatidylinositol and followed surface dilution kinetics. The interfacial Michaelis constant (Km) and the dissociation constant (Ks) for phosphatidylinositol in the Triton X-100 micelle surface were 1.3 mol % and 0.035 mM, respectively. The Km for MgATP was 0.36 mM. 55-kDa phosphatidylinositol 4-kinase catalyzed a sequential reaction mechanism as indicated by the results of kinetic and isotopic exchange reactions. The enzyme bound to phosphatidylinositol before ATP and released phosphatidylinositol 4-phosphate before ADP. The enzymological and kinetic properties of the 55-kDa phosphatidylinositol 4-kinase differed significantly from those of the 45-kDa phosphatidylinositol 4-kinase. This may suggest that the two forms of phosphatidylinositol 4-kinase from S. cerevisiae are regulated differentially in vivo.
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Phosphatidylinositol 4-kinase from yeast.
Methods in enzymology, 1992Co-Authors: George M. Carman, Charles J. Belunis, Joseph T. NickelsAbstract:Publisher Summary Phosphatidylinositol 4-kinase (1-phosphatidylinositol kinase) catalyzes the reaction of phosphatidylinositol with ATP to form phosphatidylinositol 4-phosphate. Phosphatidylinositol 4-kinase (ATP:1-phosphatidyl-lD-myo-inositol 4-phosphotransferase) is the first enzyme in the phosphorylation sequence of phosphatidylinositol leading to the formation of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate in Saccharomyces cerevisiae (S. cerevisiae). The synthesis and turnover of the polyphosphoinositides in S. cerevisiae and in higher eukaryotes play an important role in cell growth. Phosphatidylinositol 4-kinase is associated with the microsomal, plasma membrane, and cytosolic fractions of S. cerevisiae. Phosphatidylinositol 4-kinase has been purified to near homogeneity from the microsomal fraction of S. cerevisiae by standard protein purification procedures. The chapter describes the purification and properties of the enzyme. The properties of phosphatidylinositol 4-kinase are also discussed. Pure phosphatidylinositol 4-kinase can be used to synthesize radiolabeled phosphatidylinositol 4-phosphate.
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Phosphatidylinositol 4-kinase from Saccharomyces cerevisiae. Kinetic analysis using Triton X-100/phosphatidylinositol-mixed micelles.
The Journal of biological chemistry, 1991Co-Authors: Rosa J. Buxeda, Joseph T. Nickels, C J Belunis, George M. CarmanAbstract:Abstract Phosphatidylinositol 4-kinase (ATP:phosphatidylinositol 4-phosphotransferase, EC 2.7.1.67) was purified from Saccharomyces cerevisiae by an improved procedure over that previously reported (Belunis, C.J., Bae-Lee, M., Kelley, M.J., and Carman, G.M. (1988) J. Biol. Chem. 263, 18897-18903) for the enzyme. The molecular mass of the enzyme was 45 kDa. The 35-kDa protein previously identified as PI 4-kinase was a proteolysis product of the 45-kDa protein. A detailed kinetic analysis of the purified enzyme was performed with Triton X-100/phosphatidylinositol-mixed micelles according to the "surface dilution" (Deems, R.A., Eaton, B.R., and Dennis, E.A. (1975) J. Biol. Chem. 250, 9013-9020) and "dual phospholipid" (Hendrickson, H.S., and Dennis, E.A. (1984) J. Biol. Chem. 259, 5734-5739) kinetic models. Phosphatidylinositol 4-kinase activity followed saturation kinetics with respect to the bulk and surface concentrations of phosphatidylinositol at concentrations of phosphatidylinositol below 0.1 mM. Above 0.1 mM activity was only dependent on the surface concentration of phosphatidylinositol. The enzyme more closely followed the dual phospholipid model where the enzyme associated with Triton X-100 micelles when phosphatidylinositol was present. The interfacial Michaelis constant (KmB) for phosphatidylinositol was 0.0036 mol fraction and the dissociation constant (KsA) for phosphatidylinositol in the micelle surface was 0.26 mM. The results of glycerol gradient centrifugation studies showed that the enzyme was physically associated with Triton X-100/phosphatidylinositol micelles.
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Phosphatidylinositol 4-Kinase from Saccharomyces cerevisiae
1991Co-Authors: Rosa J. Buxeda, Joseph T. Nickels, Charles J. Beluniss, George M. CarmansAbstract:Phosphatidylinositol 4-kinase (ATP:phosphatidylinositol4-phosphotransferase, EC 2.7.1.67) was purified from Saccharomyces cerevisiae by an improved procedure over that previously reported (Belunis, C. J., Bae-Lee, M., Kelley, M. J., and Carman, G. M. (1988) J. Biol. Chem. 263, 18897-18903) for the enzyme. The molecular mass of the enzyme was 45 kDa. The 35-kDa protein previously identified as PI 4-kinase was a proteolysis product of the 45-kDa protein. A detailed kinetic analysis of the purified enzyme was performed with Triton X-lOO/phosphatidylinositolmixed micelles according to the “surface dilution” (Deems, R. A., Eaton, B. R., and Dennis, E. A. (1975) J. Biol. Chem. 250, 9013-9020) and “dual phospholipid” (Hendrickson, H. S., and Dennis, E. A. (1984) J. Biol. Chem. 259, 5734-5739) kinetic models. Phosphatidylinositol 4-kinase activity followed saturation kinetics with respect to the bulk and surface concentrations of phosphatidylinositol at concentrations of phosphatidylinositol below 0.1 mM. Above 0.1 mM activity was only dependent on the surface concentration of phosphatidylinositol. The enzyme more closely followed the dual phospholipid model where the enzyme associated with Triton X-100 micelles when phosphatidylinositol was present. The interfacial Michaelis constant (KmB) for phosphatidylinositol was 0.0036 mol fraction and the dissociation constant (KsA) for phosphatidylinositol in the micelle surface was 0.26 mM. The results of glycerol gradient centrifugation studies showed that the enzyme was physically associated with Triton XlOO/phosphatidylinositol micelles.
Colin T. Buckley - One of the best experts on this subject based on the ideXlab platform.
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Identification of mitogen-activated protein kinase docking sites in enzymes that metabolize Phosphatidylinositols and inositol phosphates
Cell communication and signaling : CCS, 2006Co-Authors: Kevin K. Caldwell, Marcos Sosa, Colin T. BuckleyAbstract:Background Reversible interactions between the components of cellular signaling pathways allow for the formation and dissociation of multimolecular complexes with spatial and temporal resolution and, thus, are an important means of integrating multiple signals into a coordinated cellular response. Several mechanisms that underlie these interactions have been identified, including the recognition of specific docking sites, termed a D-domain and FXFP motif, on proteins that bind mitogen-activated protein kinases (MAPKs). We recently found that phosphatidylinositol-specific phospholipase C-γ1 (PLC-γ1) directly binds to extracellular signal-regulated kinase 2 (ERK2), a MAPK, via a D-domain-dependent mechanism. In addition, we identified D-domain sequences in several other PLC isozymes. In the present studies we sought to determine whether MAPK docking sequences could be recognized in other enzymes that metabolize Phosphatidylinositols (PIs), as well as in enzymes that metabolize inositol phosphates (IPs).
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Identification of mitogen-activated protein kinase docking sites in enzymes that metabolize Phosphatidylinositols and inositol phosphates
Cell Communication and Signaling, 2006Co-Authors: Kevin K. Caldwell, Marcos Sosa, Colin T. BuckleyAbstract:Background Reversible interactions between the components of cellular signaling pathways allow for the formation and dissociation of multimolecular complexes with spatial and temporal resolution and, thus, are an important means of integrating multiple signals into a coordinated cellular response. Several mechanisms that underlie these interactions have been identified, including the recognition of specific docking sites, termed a D-domain and FXFP motif, on proteins that bind mitogen-activated protein kinases (MAPKs). We recently found that phosphatidylinositol-specific phospholipase C-γ1 (PLC-γ1) directly binds to extracellular signal-regulated kinase 2 (ERK2), a MAPK, via a D-domain-dependent mechanism. In addition, we identified D-domain sequences in several other PLC isozymes. In the present studies we sought to determine whether MAPK docking sequences could be recognized in other enzymes that metabolize Phosphatidylinositols (PIs), as well as in enzymes that metabolize inositol phosphates (IPs). Results We found that several, but not all, of these enzymes contain identifiable D-domain sequences. Further, we found a high degree of conservation of these sequences and their location in human and mouse proteins; notable exceptions were PI 3-kinase C2-γ, PI 4-kinase type IIβ, and inositol polyphosphate 1-phosphatase. Conclusion The results indicate that there may be extensive crosstalk between MAPK signaling and signaling pathways that are regulated by cellular levels of PIs or IPs.
Gordon B Mills - One of the best experts on this subject based on the ideXlab platform.
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src family protein tyrosine kinases alter the function of pten to regulate phosphatidylinositol 3 kinase akt cascades
Journal of Biological Chemistry, 2003Co-Authors: Jue Hui Liu, Jinyi Zhang, Hongwei Wang, Dimpy Koul, John S Mcmurray, Xianjun Fang, W Alfred K Yung, Kathy Siminovitch, Gordon B MillsAbstract:Src family protein-tyrosine kinases, which play an important role in signal integration, have been implicated in tumorigenesis in multiple lineages, including breast cancer. We demonstrate, herein, that Src kinases regulate the phosphatidylinositol 3-kinase (PI3K) signaling cascade via altering the function of the PTEN tumor suppressor. Overexpression of activated Src protein-tyrosine kinases in PTEN-deficient breast cancer cells does not alter AKT phosphorylation, an indicator of signal transduction through the PI3K pathway. However, in the presence of functional PTEN, Src reverses the activity of PTEN, resulting in an increase in AKT phosphorylation. Activated Src reduces the ability of PTEN to dephosphorylate Phosphatidylinositols in micelles and promotes AKT translocation to cellular plasma membranes but does not alter PTEN activity toward water-soluble Phosphatidylinositols. Thus, Src may alter the capacity of the PTEN C2 domain to bind cellular membranes rather than directly interfering with PTEN enzymatic activity. Tyrosine phosphorylation of PTEN is increased in breast cancer cells treated with pervanadate, suggesting that PTEN contains sites for tyrosine phosphorylation. Src kinase inhibitors markedly decreased pervanadate-mediated tyrosine phosphorylation of PTEN. Further, expression of activated Src results in marked tyrosine phosphorylation of PTEN. SHP-1, a SH2 domain-containing protein-tyrosine phosphatase, selectively binds and dephosphorylates PTEN in Src transfected cells. Both Src inhibitors and SHP-1 overexpression reverse Src-induced loss of PTEN function. Coexpression of PTEN with activated Src reduces the stability of PTEN. Taken together, the data indicate that activated Src inhibits PTEN function leading to alterations in signaling through the PI3K/AKT pathway.