Control of Liver Glycogen Synthase Activity and Intracellular Distribution by Phosphorylation

Eukaryotic glycogen synthase activity is regulated by reversible phosphorylation at multiple sites. Of the two GS isoforms found in mammals, the muscle enzyme (muscle glycogen synthase) has received more attention and the relative importance of every known phosphorylation site in the control of its activity and intracellular distribution has been previously addressed. We have analyzed the impact of the dephosphorylation at the homologous sites of the glycogen synthase liver (LGS) isoform. Serine residues at these sites were replaced by non-phosphorylatable alanine residues, singly or in pairs, and the resultant LGS variants were expressed in cultured cells using adenoviral vectors. The sole mutation at site 2 (Ser7) yielded an enzyme that was almost fully active and able to induce glycogen deposition in primary hepatocytes incubated in the absence of glucose and in FTO2B cells, a cell line that does not normally synthesize glycogen. Mutation at site 2 was also sufficient to trigger the aggregation and translocation of LGS from the cytoplasm to the hepatocyte cell cortex in the absence of glucose. However, this redistribution was not observed in hepatocytes incubated without glucose when an additional mutation (E509A), which renders the enzyme inactive, was introduced. This result suggests that LGS translocation is strictly dependent on glycogen synthesis. Eukaryotic glycogen synthase activity is regulated by reversible phosphorylation at multiple sites. Of the two GS isoforms found in mammals, the muscle enzyme (muscle glycogen synthase) has received more attention and the relative importance of every known phosphorylation site in the control of its activity and intracellular distribution has been previously addressed. We have analyzed the impact of the dephosphorylation at the homologous sites of the glycogen synthase liver (LGS) isoform. Serine residues at these sites were replaced by non-phosphorylatable alanine residues, singly or in pairs, and the resultant LGS variants were expressed in cultured cells using adenoviral vectors. The sole mutation at site 2 (Ser7) yielded an enzyme that was almost fully active and able to induce glycogen deposition in primary hepatocytes incubated in the absence of glucose and in FTO2B cells, a cell line that does not normally synthesize glycogen. Mutation at site 2 was also sufficient to trigger the aggregation and translocation of LGS from the cytoplasm to the hepatocyte cell cortex in the absence of glucose. However, this redistribution was not observed in hepatocytes incubated without glucose when an additional mutation (E509A), which renders the enzyme inactive, was introduced. This result suggests that LGS translocation is strictly dependent on glycogen synthesis. The rate-limiting enzyme for glycogen synthesis is glycogen synthase (GS), 3The abbreviations used are: GS, glycogen synthase; DMEM, Dulbecco's modified Eagle's medium; LGS, liver glycogen synthase; MGS, muscle glycogen synthase; GSK-3, glycogen synthase kinase 3 GSK-3; PTG, protein targeting to glycogen; FBS, fetal bovine serum; TRITC, tetramethylrhodamine isothiocyanate. which catalyzes the addition of α1,4-linked glucose units from UDP-glucose to a growing glycogen chain. The activity of the enzyme is tightly regulated by phosphorylation at multiple sites and by allosteric effectors, such that phosphorylation tends to inactivate the enzyme, but even the most phosphorylated forms become fully active in the presence of glucose-6-P (Glc-6-P). In mammals there are two GS isoforms, liver glycogen synthase (LGS), whose expression is tissue-specific, and muscle glycogen synthase (MGS), which is expressed in almost all tissues (1Roach P.J. Cheng C. Huang D. Lin A. Mu J. Skurat A.V. Wilson W. Zhai L. J. Basic Clin. Physiol. Pharmacol. 1998; 9: 139-151Crossref PubMed Scopus (44) Google Scholar). Although the two isoforms are 70% identical, the NH2- and COOH-terminal extremes are only 50% homologous (2Bai G. Zhang Z.J. Werner R. Nuttall F.Q. Tan A.W. Lee E.Y. J. Biol. Chem. 1990; 265: 7843-7848Abstract Full Text PDF PubMed Google Scholar), and the LGS COOH-terminal domain is shorter (3Hanashiro I. Roach P.J. Arch. Biochem. Biophys. 2002; 397: 286-292Crossref PubMed Scopus (31) Google Scholar). The regulatory sites of MGS that undergo reversible phosphorylation have been identified: two of them, sites 2 and 2a, are located near the NH2 terminus, whereas the remaining seven sites, 3a, 3b, 3c, 4, 5, 1a, and 1b, are found within the COOH-terminal 100 residues (Fig. 1). The relative importance of each site in the regulation of MGS activity has been documented (4Skurat A.V. Wang Y. Roach P.J. J. Biol. Chem. 1994; 269: 25534-25542Abstract Full Text PDF PubMed Google Scholar, 5Skurat A.V. Roach P.J. J. Biol. Chem. 1995; 270: 12491-12497Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 6Skurat A.V. Roach P.J. Biochem. J. 1996; 313: 45-50Crossref PubMed Scopus (27) Google Scholar). A similar type of analysis has not been performed with LGS, in which homologues of seven of the nine MGS phosphorylation sites are present (Fig. 1). Only one mutant form of LGS, which contains six Ser to Ala substitutions at sites 2, 3a, 3b, 3c, 4, and 5, has been studied. This mutant is constitutively active and causes a large increase in glycogen accumulation when expressed in rat primary hepatocytes (7Kadotani A. Fujimura M. Nakamura T. Ohyama S. Harada N. Maruki H. Tamai Y. Kanatani A. Eiki J. Nagata Y. Arch. Biochem. Biophys. 2007; 466: 283-289Crossref PubMed Scopus (6) Google Scholar). LGS is phosphorylated at multiple sites when hepatocytes are incubated with glycogenolytic agents such as adrenaline, glucagon, or phorbol esters (8Arino J. Mor A. Bosch F. Baanante I.V. Guinovart J.J. FEBS Lett. 1984; 170: 310-314Crossref PubMed Scopus (11) Google Scholar, 9Arino J. Guinovart J.J. Biochem. Biophys. Res. Commun. 1986; 134: 113-119Crossref PubMed Scopus (8) Google Scholar). Furthermore, LGS can be phosphorylated in vitro by several kinases, including cAMP-dependent protein kinase, protein kinase C, casein kinase 1 and 2, AMP-stimulated protein kinase, phosphorylase kinase, and glycogen synthase kinase 3 (GSK-3) (10Roach P.J. FASEB J. 1990; 4: 2961-2968Crossref PubMed Scopus (194) Google Scholar, 11Roach P.J. Bayer P.D. Krebs E.G. Enzymes. 3rd eds. vol 17. Academic Press, New York1986: 499-539Google Scholar). The muscle and liver isoforms of GS also differ in their intracellular distribution. When glycogen stores are depleted in cells incubated in the absence of glucose, MGS concentrates in the nucleus, but moves to the cytoplasm, where glycogen deposition occurs, when levels of the monosaccharide increase (12Ferrer J.C. Baque S. Guinovart J.J. FEBS Lett. 1997; 415: 249-252Crossref PubMed Scopus (49) Google Scholar, 13Cid E. Gomis R.R. Geremia R.A. Guinovart J.J. Ferrer J.C. J. Biol. Chem. 2000; 275: 33614-33621Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). An arginine-rich cluster, located near the COOH terminus and involved in the MGS allosteric activation by Glc-6-P (3Hanashiro I. Roach P.J. Arch. Biochem. Biophys. 2002; 397: 286-292Crossref PubMed Scopus (31) Google Scholar), is also required for the nuclear accumulation of the enzyme (14Cid E. Cifuentes D. Baque S. Ferrer J.C. Guinovart J.J. FEBS J. 2005; 272: 3197-3213Crossref PubMed Scopus (21) Google Scholar). However, the regulation of the nucleocytoplasmic shuttling does not reside in any of the known MGS phosphorylation sites that control its activity (14Cid E. Cifuentes D. Baque S. Ferrer J.C. Guinovart J.J. FEBS J. 2005; 272: 3197-3213Crossref PubMed Scopus (21) Google Scholar). In sharp contrast, LGS translocates from a diffuse cytoplasmic distribution to the cell cortex in response to glucose (15Fernandez-Novell J.M. Bellido D. Vilaro S. Guinovart J.J. Biochem. J. 1997; 321: 227-231Crossref PubMed Scopus (66) Google Scholar, 16Garcia-Rocha M. Roca A. De La Iglesia N. Baba O. Fernandez-Novell J.M. Ferrer J.C. Guinovart J.J. Biochem. J. 2001; 357: 17-24Crossref PubMed Scopus (73) Google Scholar). Consequently, in glycogen synthesis is at the and the J.M. C. Ferrer J.C. Guinovart J.J. FEBS Lett. 2002; PubMed Scopus Google Scholar). the relative of each of the LGS phosphorylation sites in the regulation of its We also analyzed the phosphorylation of these sites the intracellular distribution of the liver in to with MGS, whose activation dephosphorylation of site 2, at the NH2 terminus, and site or 3b, at the COOH terminus (4Skurat A.V. Wang Y. Roach P.J. J. Biol. Chem. 1994; 269: 25534-25542Abstract Full Text PDF PubMed Google Scholar), a Ser to Ala mutation at site 2 of LGS is sufficient to an fully active form of the liver of for liver GS has been R.R. Ferrer J.C. Guinovart J.J. Biochem. J. 2000; PubMed Scopus Google Scholar). Ser to Ala were at sites 2, 2a, 3a, 3b, 3c, 4, and by using the site 2 and site and site and site and site and site and and site and The for LGS in these sites or in of two were as T. Biol. 1994; PubMed Scopus Google Scholar). The mutation was and 2 using the and A for the enzyme was used as a control in the impact The the were using the and an and with was used to hepatocytes from for as previously J. Guinovart J.J. FEBS Lett. PubMed Scopus Google Scholar). were in modified with glucose, fetal bovine 100 and 100 and of with at a of were replaced with glucose and FBS, and cells were for 2 with at a similar of were replaced by without glucose or FBS, and of was were incubated in in the absence or presence of glucose, as in the and the of each cell were in and at FTO2B and with FTO2B rat cells I. S. 1998; PubMed Scopus Google were cultured in and in with glucose and were replaced with glucose and FBS, and cells were for 2 with at the of this were replaced with glucose and FBS, and cells were incubated for at were replaced by without glucose or FBS, and of was were incubated in glucose, as in the and the of each cell were in and at and GS activity in cell cell in were using 100 of of 1 1 and The were a was the of Biochem. PubMed Scopus Google using a GS activity was in or in the and of hepatocyte at for in the presence or absence of Glc-6-P J. Biochem. PubMed Scopus Google Scholar). GS activity in the presence of Glc-6-P on the of enzyme, whereas in the absence of Glc-6-P is an of the active GS The Glc-6-P activity is an of the activation of the was using an as J. Biol. Chem. Full Text PDF PubMed Google Scholar). and of protein of the GS activity was by a and with the a rat LGS M. Roca A. De La Iglesia N. Baba O. Fernandez-Novell J.M. Ferrer J.C. Guinovart J.J. Biochem. J. 2001; 357: 17-24Crossref PubMed Scopus (73) Google Scholar), a GS phosphorylated on or a We also used a GS phosphorylated on site The was in the which residues of rat LGS to or were were using an the were using on were for in cells were incubated with 1 for and for with of with bovine with the primary and was were and using We used primary rat LGS M. Roca A. De La Iglesia N. Baba O. Fernandez-Novell J.M. Ferrer J.C. Guinovart J.J. Biochem. J. 2001; 357: 17-24Crossref PubMed Scopus (73) Google and a glycogen O. PubMed Scopus Google from O. and The used were and tetramethylrhodamine are expressed as the was by the using was at of the Mutation of the on LGS the phosphorylation sites that LGS seven adenoviral for mutant forms of rat In each mutant one of the seven Ser residues homologous to the MGS phosphorylation sites (Fig. was replaced by an Ala The residues were and and the mutant forms are to as LGS 2, 2a, 3a, 3b, 3c, 4, or 5, and when primary hepatocytes were with these or with control from or rat LGS glucose the dephosphorylation and activation of LGS H. J. Biochem. PubMed Scopus Google Scholar, H. W. J. Biochem. PubMed Scopus Google Scholar, J. S. Biochem. J. PubMed Scopus Google Scholar), all were performed in the absence of the monosaccharide to dephosphorylation of the Ser residues that were not GS activity in cell that similar of the LGS and mutant forms were expressed in all (Fig. We the GS activity in the and of the hepatocyte the (Fig. The GS activity of the as in the control cells that (Fig. is with the activity found in the hepatocyte LGS or the phosphorylation the activity can be to each mutant without any Although at sites 2a, 3c, 4, and not a in the activation of the enzyme, the LGS and LGS variants a increase of the activity and the mutation at site 2 in a large increase of this to in the and We the activation by dephosphorylation of site 2 be by the dephosphorylation of a The LGS variants that Ser to Ala substitutions at site 2 an additional site activity the were not when with LGS of LGS in FTO2B cell FTO2B cells the most I. S. 1998; PubMed Scopus Google Scholar). Although this cell line LGS, the enzyme is in a phosphorylated and these cells are to synthesize glycogen when incubated with glucose. deposition can be the expression of A. Bosch F. J. Biochem. 1994; PubMed Scopus Google Scholar, R.R. E. M. Ferrer J.C. Guinovart J.J. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). The intracellular levels of Glc-6-P LGS allosteric activation but also its dephosphorylation by C. Guinovart J.J. FASEB J. 1997; PubMed Scopus Google Scholar). its of the FTO2B cell line is an to the activation of the LGS phosphorylation and their to glycogen deposition in the presence of glucose, without the dephosphorylation of LGS sites. FTO2B cells were with LGS or the mutant forms and were incubated in the presence of glucose. of GS activity that LGS and the mutant forms were expressed to similar levels (Fig. The activity in the of FTO2B cells LGS with the LGS or the LGS 3a, 3b, or variants were or whereas the of cells LGS 2, or the at this site one additional activity (Fig. in the performed with the the activity of the LGS 2 mutant and the were not be GS activity of the FTO2B cells was not with the activity of the cells LGS or the mutant forms from to of the GS the activity in the FTO2B are an of the of the mutant forms with activity that the activity of for LGS in control FTO2B cells with the (Fig. does not in cells LGS 2, the for this mutant is with the of the LGS phosphorylation to trigger glycogen deposition in FTO2B LGS and the 3a, 3b, and variants levels of similar to found in control cells whereas the and the Ser to Ala mutation at site 2 yielded levels of the (Fig. of the We analyzed the Ser to Ala of the regulatory sites any on the phosphorylation of sites 2 and of LGS expressed in that LGS or LGS phosphorylated site 2 performed analysis of hepatocytes LGS or the LGS dephosphorylation of any of the sites not a in the of site 2 (Fig. A similar analysis was performed using an that LGS only when site is phosphorylated In this for the LGS there was a in phosphorylation at site 3a, whereas the remaining LGS were all phosphorylated at this site (Fig. LGS 2 and LGS were not by the that the and also observed for MGS (4Skurat A.V. Wang Y. Roach P.J. J. Biol. Chem. 1994; 269: 25534-25542Abstract Full Text PDF PubMed Google Scholar), the LGS in when sites were Only the of LGS variants in which sites or were (Fig. This the that Ser to Ala at the NH2- or COOH-terminal regulatory sites of LGS not phosphorylation of sites and of the Mutation of the on the of of hepatocytes with glucose LGS and also causes its translocation from a cytoplasmic distribution to the cell (15Fernandez-Novell J.M. Bellido D. Vilaro S. Guinovart J.J. Biochem. J. 1997; 321: 227-231Crossref PubMed Scopus (66) Google Scholar, 16Garcia-Rocha M. Roca A. De La Iglesia N. Baba O. Fernandez-Novell J.M. Ferrer J.C. Guinovart J.J. Biochem. J. 2001; 357: 17-24Crossref PubMed Scopus (73) Google Scholar, J.M. C. Ferrer J.C. Guinovart J.J. FEBS Lett. 2002; PubMed Scopus Google Scholar). the impact of the phosphorylation of the regulatory sites on LGS performed on hepatocytes the LGS the control hepatocytes with the was that LGS was not LGS an enzyme the cytoplasm in the absence of glucose, as for LGS (15Fernandez-Novell J.M. Bellido D. Vilaro S. Guinovart J.J. Biochem. J. 1997; 321: 227-231Crossref PubMed Scopus (66) Google Scholar, 16Garcia-Rocha M. Roca A. De La Iglesia N. Baba O. Fernandez-Novell J.M. Ferrer J.C. Guinovart J.J. Biochem. J. 2001; 357: 17-24Crossref PubMed Scopus (73) Google Scholar). However, LGS 2 the of a enzyme at the of glycogen even when the hepatocytes were incubated in the absence of glucose. The remaining LGS LGS (Fig. The Ser to Ala at site 2 one of the six regulatory sites also in the absence of glucose, in the form of near the hepatocyte cortex not the by the LGS 2 mutant was of hepatocytes this mutant was able to the deposition of the in hepatocytes incubated without glucose. analysis with an O. PubMed Scopus Google that whereas hepatocytes LGS or any of the not glycogen in the absence of glucose, cells LGS 2 (Fig. or LGS 2 not synthesize even in these of primary rat hepatocytes site phosphorylation of were for analysis with an glycogen. of cultured for in the absence of glucose, LGS or Ser to Ala LGS at the phosphorylation sites. were using a with an additional The translocation be from glycogen synthesis. this for the variants LGS and LGS 2 We have previously that in MGS the of by an Ala an enzyme E. Gomis R.R. Geremia R.A. Guinovart J.J. Ferrer J.C. J. Biol. Chem. 2000; 275: 33614-33621Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). with these expressed similar of the mutant as by analysis (Fig. The of control hepatocytes with the and of cells LGS and LGS 2 GS activity (Fig. that the of LGS was by the were found in the glycogen of LGS LGS 2 and cells (Fig. analysis of the intracellular distribution of LGS and LGS 2 in cultured hepatocytes in the presence and absence of glucose, the two a distribution to the enzyme (Fig. In the absence of glucose the cytoplasm, but in the cell cortex when the monosaccharide was to glycogen by the analysis of of LGS expressed in hepatocytes were for analysis with an of cells LGS LGS 2 3b, and the LGS and LGS 2 were cultured for in the absence or presence of glucose was that LGS was not were using a with an additional The Although the activity of the two isoforms of glycogen MGS and LGS, is by reversible phosphorylation and allosteric effectors, the of the regulation differ the two MGS, the activation of Ser to Ala variants at the phosphorylation sites was not to that of the enzyme, when was to the NH2 or COOH terminus (4Skurat A.V. Wang Y. Roach P.J. J. Biol. Chem. 1994; 269: 25534-25542Abstract Full Text PDF PubMed Google Scholar). An MGS mutant at site 2 an activity of only and an additional Ser to Ala mutation at site or was required to increase this (4Skurat A.V. Wang Y. Roach P.J. J. Biol. Chem. 1994; 269: 25534-25542Abstract Full Text PDF PubMed Google Scholar). In contrast, the sole dephosphorylation of LGS at site 2 its activity to in hepatocytes and FTO2B This is to that previously found for an LGS mutant six Ser to Ala substitutions at sites 2, 3a, 3b, 3c, 4, and 5, which an activity of (7Kadotani A. Fujimura M. Nakamura T. Ohyama S. Harada N. Maruki H. Tamai Y. Kanatani A. Eiki J. Nagata Y. Arch. Biochem. Biophys. 2007; 466: 283-289Crossref PubMed Scopus (6) Google Scholar). that the LGS 2 mutant is fully active and that most of the control by phosphorylation on LGS activity in site 2 The MGS with six Ser to Ala substitutions at the homologous sites an activity of A.V. Roach P.J. J. Biol. Chem. 1995; 270: 12491-12497Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). This the MGS and LGS, as that the of Glc-6-P to increase the activity of a fully enzyme is in the liver in the muscle of The of a that at the phosphorylation and Glc-6-P the activity of these to these at this An for the phosphorylation of the COOH-terminal sites of GS has been previously (10Roach P.J. FASEB J. 1990; 4: 2961-2968Crossref PubMed Scopus (194) Google Scholar, 11Roach P.J. Bayer P.D. Krebs E.G. Enzymes. 3rd eds. vol 17. Academic Press, New York1986: 499-539Google Scholar, C. J. FEBS Lett. PubMed Scopus Google Scholar). to this phosphorylation at sites 3a, 3b, 3c, 4, and in a with the of a at site by the of at the sites. not this and that mutation of COOH-terminal sites 3c, 4, or does not phosphorylation at site of the LGS expressed in cultured Only the of site has on the to which site is were for MGS, which can be phosphorylated at sites and in cells or even when the is by Ser to Ala at sites 3c, 4, or (4Skurat A.V. Wang Y. Roach P.J. J. Biol. Chem. 1994; 269: 25534-25542Abstract Full Text PDF PubMed Google Scholar, 5Skurat A.V. Roach P.J. J. Biol. Chem. 1995; 270: 12491-12497Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 6Skurat A.V. Roach P.J. Biochem. J. 1996; 313: 45-50Crossref PubMed Scopus (27) Google Scholar, Skurat A.V. A. Roach P.J. J. S. A. 2005; PubMed Scopus Google Scholar). We that additional GSK-3, in hepatocytes for the COOH-terminal phosphorylation of also a the of the phosphorylation sites located at the COOH terminus of LGS, or on the activation of this be that these sites have the regulation of LGS activity that have not been However, be that the used in this and with MGS (4Skurat A.V. Wang Y. Roach P.J. J. Biol. Chem. 1994; 269: 25534-25542Abstract Full Text PDF PubMed Google Scholar, 6Skurat A.V. Roach P.J. Biochem. J. 1996; 313: 45-50Crossref PubMed Scopus (27) Google to the of each regulatory the of Ser residues by non-phosphorylatable Ala residues, in the of the enzyme to The is to be when a large of LGS the of a site not all the from one to in have P.J. N. J. Biochem. PubMed Scopus Google Scholar, P.J. J. Biochem. PubMed Scopus Google Scholar, L. J. J. Biochem. PubMed Scopus Google Scholar, S. J. Biochem. PubMed Scopus Google Scholar), these are almost and activation or of LGS is the result of more in the of the In this which for a of LGS activity in response to the phosphorylation sites at the COOH terminus be in the control of the LGS activation The the of a of GSK-3, in this S. A. 1996; PubMed Scopus Google Scholar). of cultured hepatocytes with a and increase in the LGS activity F. J. Guinovart J.J. J. Biol. Chem. 1986; Full Text PDF PubMed Google Scholar), a of of M. Roca A. De La Iglesia N. Baba O. Fernandez-Novell J.M. Ferrer J.C. Guinovart J.J. Biochem. J. 2001; 357: 17-24Crossref PubMed Scopus (73) Google Scholar). of this is that the activation of LGS, a Ser to Ala mutation at site 2, an enzyme that is able to glycogen deposition in in the absence of glucose, and in FTO2B The cell line all the to synthesize with the only This enzyme is to Glc-6-P at the required levels or in the R.R. E. M. Ferrer J.C. Guinovart J.J. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, J. C. Guinovart J.J. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google to active LGS trigger glycogen synthesis in response to glucose A. Bosch F. I. Guinovart J.J. Arch. Biochem. Biophys. PubMed Scopus (21) Google Scholar). In FTO2B cells, the LGS 2 mutant this by of its constitutively activation The for the cultured hepatocytes is more glycogen even when are incubated in the absence of glucose. The glucose units the glycogen from present in the contains and a of the LGS with six Ser to Ala at sites 2, 3a, 3b, 3c, 4, and were to large of using the glucose the even when incubated in the presence of glucose (7Kadotani A. Fujimura M. Nakamura T. Ohyama S. Harada N. Maruki H. Tamai Y. Kanatani A. Eiki J. Nagata Y. Arch. Biochem. Biophys. 2007; 466: 283-289Crossref PubMed Scopus (6) Google Scholar). The of cultured hepatocytes J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google and of D. J. Clin. 2000; PubMed Scopus Google to glycogen was also when the protein targeting to glycogen in these the was the for the synthesis of the in the that has a impact on the glycogen as a and for from the activation of LGS, also a in glycogen phosphorylase activity J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). In contrast, LGS 2 and the glycogen accumulation in a in which liver glycogen phosphorylase be of the absence of glucose in the When hepatocytes are incubated with glucose, LGS moves from the cytoplasm to the cell where forms the of which with (15Fernandez-Novell J.M. Bellido D. Vilaro S. Guinovart J.J. Biochem. J. 1997; 321: 227-231Crossref PubMed Scopus (66) Google Scholar, 16Garcia-Rocha M. Roca A. De La Iglesia N. Baba O. Fernandez-Novell J.M. Ferrer J.C. Guinovart J.J. Biochem. J. 2001; 357: 17-24Crossref PubMed Scopus (73) Google Scholar). Although glycogen is LGS intracellular distribution that of the that LGS to its and in the of the hepatocyte M. Roca A. De La Iglesia N. Baba O. Fernandez-Novell J.M. Ferrer J.C. Guinovart J.J. Biochem. J. 2001; 357: 17-24Crossref PubMed Scopus (73) Google Scholar). We have also that when glycogen stores are the sites in which synthesis of the are located in the hepatocyte However, as glycogen deposition more sites are from the cortex to the hepatocyte J.M. C. Ferrer J.C. Guinovart J.J. FEBS Lett. 2002; PubMed Scopus Google Scholar). that dephosphorylation at site 2 of LGS causes the accumulation of the enzyme at the hepatocyte even in the absence of glucose. In contrast, the LGS 2 only in the presence of the We that this is a of the of LGS to glycogen the dephosphorylation of site 2 a for the translocation of the the constitutively active LGS 2 mutant to the sites even in the absence of glucose, the synthesis of which in more LGS to the aggregation and accumulation of the enzyme observed at the hepatocyte The LGS 2 mutant also glycogen by the LGS in the presence of glucose. However, this glycogen does not sites to which LGS can when glucose is from the in this is the that the activation of LGS, phosphorylation sites site 2, does not glycogen synthesis LGS translocation in hepatocytes incubated without glucose M. Roca A. De La Iglesia N. Baba O. Fernandez-Novell J.M. Ferrer J.C. Guinovart J.J. Biochem. J. 2001; 357: 17-24Crossref PubMed Scopus (73) Google Scholar, F. J. Guinovart J.J. J. Biol. Chem. 1986; Full Text PDF PubMed Google Scholar). This result also as as there is glycogen activation of LGS does not to its In site 2 of LGS (Ser7) is the most regulatory site of the activity of the which an almost fully active enzyme, is not sufficient to trigger LGS translocation to the hepatocyte and glycogen accumulation is also required to this We R. from the and of for in the of the We R. R. Gomis for the LGS We are also to A. M. C. and E. for and T. for the of the