共查询到20条相似文献,搜索用时 15 毫秒
1.
Shiota C Coffey J Grimsby J Grippo JF Magnuson MA 《The Journal of biological chemistry》1999,274(52):37125-37130
Hepatic glucokinase (GK) moves between the nucleus and cytoplasm in response to metabolic alterations. Here, using heterologous cell systems, we have found that at least two different mechanisms are involved in the intracellular movement of GK. In the absence of the GK regulatory protein (GKRP) GK resides only in the cytoplasm. However, in the presence of GKRP, GK moves to the nucleus and resides there in association with this protein until changes in the metabolic milieu prompt its release. GK does not contain a nuclear localization signal sequence and does not enter the nucleus in a GKRP-independent manner because cells treated with leptomycin B, a specific inhibitor of leucine-rich NES-dependent nuclear export, do not accumulate GK in the nucleus. Instead, entry of GK into the nucleus appears to occur via a piggy-back mechanism that involves binding to GKRP. Nuclear export of GK, which occurs after its release from GKRP, is due to a leucine-rich nuclear export signal within the protein ((300)ELVRLVLLKLV(310)). Thus, GKRP appears to function as both a nuclear chaperone and metabolic sensor and is a critical component of a hepatic GK translocation cycle for regulating the activity of this enzyme in response to metabolic alterations. 相似文献
2.
Thomas L. Jetton Masa Shiota Susan M. Knobel David W. Piston Alan D. Cherrington Mark A. Magnuson 《Experimental diabetes research》2001,2(3):173-186
Hepatic glucokinase (GK) is acutely regulated by
binding to its nuclear-anchored regulatory protein
(GKRP). Although GK release by GKRP is tightly
coupled to the rate of glycogen synthesis, the nature
of this association is obscure. To gain insight into
this coupling mechanism under physiological stimulating
conditions in primary rat hepatocytes, we
analyzed the subcellular distribution of GK and
GKRP with immunofluorescence, and glycogen deposition
with glycogen cytochemical fluorescence,
using confocal microscopyand quantitative image
analysis. Following stimulation, a fraction of the GK
signal translocated from the nucleus to the cytoplasm.
The reduction in the nuclear to cytoplasmic
ratio of GK, an index of nuclear export, correlated
with a >50% increase in glycogen cytochemical
fluorescence over a 60min stimulation period. Furthermore,
glycogen accumulation was initially deposited
in a peripheral pattern in hepatocytes
similar to that of GK. These data suggest that a compartmentalization
exists of both active GK and the
initial sites of glycogen deposition at the hepatocyte
surface. 相似文献
3.
Mukhtar MH Payne VA Arden C Harbottle A Khan S Lange AJ Agius L 《American journal of physiology. Regulatory, integrative and comparative physiology》2008,294(3):R766-R774
The rate of glucose phosphorylation in hepatocytes is determined by the subcellular location of glucokinase and by its association with its regulatory protein (GKRP) in the nucleus. Elevated glucose concentrations and precursors of fructose 1-phosphate (e.g., sorbitol) cause dissociation of glucokinase from GKRP and translocation to the cytoplasm. In this study, we investigated the counter-regulation of substrate-induced translocation by AICAR (5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside), which is metabolized by hepatocytes to an AMP analog, and causes activation of AMP-activated protein kinase (AMPK) and depletion of ATP. During incubation of hepatocytes with 25 mM glucose, AICAR concentrations below 200 microM activated AMPK without depleting ATP and inhibited glucose phosphorylation and glucokinase translocation with half-maximal effect at 100-140 microM. Glucose phosphorylation and glucokinase translocation correlated inversely with AMPK activity. AICAR also counteracted translocation induced by a glucokinase activator and partially counteracted translocation by sorbitol. However, AICAR did not block the reversal of translocation (from cytoplasm to nucleus) after substrate withdrawal. Inhibition of glucose-induced translocation by AICAR was greater than inhibition by glucagon and was associated with phosphorylation of both GKRP and the cytoplasmic glucokinase binding protein, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK2) on ser-32. Expression of a kinase-active PFK2 variant lacking ser-32 partially reversed the inhibition of translocation by AICAR. Phosphorylation of GKRP by AMPK partially counteracted its inhibitory effect on glucokinase activity, suggesting altered interaction of glucokinase and GKRP. In summary, mechanisms downstream of AMPK activation, involving phosphorylation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase and GKRP are involved in the ATP-independent inhibition of glucose-induced glucokinase translocation by AICAR in hepatocytes. 相似文献
4.
J M Fernández-Novell S Castel D Bellido J C Ferrer S Vilaró J J Guinovart 《FEBS letters》1999,459(2):211-214
We have studied the intracellular distribution in vivo of glucokinase (GK) and glucokinase regulatory protein (GKRP) in livers of fasted and refed rats, using specific antibodies against both proteins and laser confocal fluorescence microscopy. GK was found predominantly in the nucleus of hepatocytes from starved rats. GK was translocated to the cytoplasm in livers of 1- and 2-h refed animals, but returned to the nucleus after 4 h. GKRP concentrated in the hepatocyte nuclei and its distribution did not change upon refeeding. These results show that, in physiological conditions, GKRP is present predominantly in the nuclei of hepatocytes and that the translocation of hepatic GK from and to the nucleus is operative in vivo. 相似文献
5.
The role of the regulatory protein of glucokinase in the glucose sensory mechanism of the hepatocyte 总被引:6,自引:0,他引:6
de la Iglesia N Mukhtar M Seoane J Guinovart JJ Agius L 《The Journal of biological chemistry》2000,275(14):10597-10603
Glucokinase has a very high flux control coefficient (greater than unity) on glycogen synthesis from glucose in hepatocytes (Agius et al., J. Biol. Chem. 271, 30479-30486, 1996). Hepatic glucokinase is inhibited by a 68-kDa glucokinase regulatory protein (GKRP) that is expressed in molar excess. To establish the relative control exerted by glucokinase and GKRP, we applied metabolic control analysis to determine the flux control coefficient of GKRP on glucose metabolism in hepatocytes. Adenovirus-mediated overexpression of GKRP (by up to 2-fold above endogenous levels) increased glucokinase binding and inhibited glucose phosphorylation, glycolysis, and glycogen synthesis over a wide range of concentrations of glucose and sorbitol. It decreased the affinity of glucokinase translocation for glucose and increased the control coefficient of glucokinase on glycogen synthesis. GKRP had a negative control coefficient of glycogen synthesis that is slightly greater than unity (-1.2) and a control coefficient on glycolysis of -0.5. The control coefficient of GKRP on glycogen synthesis decreased with increasing glucokinase overexpression (4-fold) at elevated glucose concentration (35 mM), which favors dissociation of glucokinase from GKRP, but not at 7.5 mM glucose. Under the latter conditions, glucokinase and GKRP have large and inverse control coefficients on glycogen synthesis, suggesting that a large component of the positive control coefficient of glucokinase is counterbalanced by the negative coefficient of GKRP. It is concluded that glucokinase and GKRP exert reciprocal control; therefore, mutations in GKRP affecting the expression or function of the protein may impact the phenotype even in the heterozygote state, similar to glucokinase mutations in maturity onset diabetes of the young type 2. Our results show that the mechanism comprising glucokinase and GKRP confers a markedly extended responsiveness and sensitivity to changes in glucose concentration on the hepatocyte. 相似文献
6.
《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》2014,1843(6):1123-1134
Glucokinase activity is a major determinant of hepatic glucose metabolism and blood glucose homeostasis. Liver glucokinase activity is regulated acutely by adaptive translocation between the nucleus and the cytoplasm through binding and dissociation from its regulatory protein (GKRP) in the nucleus. Whilst the effect of glucose on this mechanism is well established, the role of hormones in regulating glucokinase location and its interaction with binding proteins remains unsettled. Here we show that treatment of rat hepatocytes with 25 mM glucose caused decreased binding of glucokinase to GKRP, translocation from the nucleus and increased binding to 6-phosphofructo 2-kinase/fructose 2,6 bisphosphatase-2 (PFK2/FBPase2) in the cytoplasm. Glucagon caused dissociation of glucokinase from PFK2/FBPase2, concomitant with phosphorylation of PFK2/FBPase2 on Ser-32, uptake of glucokinase into the nucleus and increased interaction with GKRP. Two novel glucagon receptor antagonists attenuated the action of glucagon. This establishes an unequivocal role for hormonal control of glucokinase translocation. Given that glucagon excess contributes to the pathogenesis of diabetes, glucagon may play a role in the defect in glucokinase translocation and activity evident in animal models and human diabetes. 相似文献
7.
Martin Tobias Kaminski Julia Schultz Rica Waterstradt Markus Tiedge Sigurd Lenzen Simone Baltrusch 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》2014
The glucose phosphorylating enzyme glucokinase regulates glucose metabolism in the liver. Glucokinase activity is modulated by a liver-specific competitive inhibitor, the glucokinase regulatory protein (GRP), which mediates sequestration of glucokinase to the nucleus at low glucose concentrations. However, the mechanism of glucokinase nuclear export is not fully understood. In this study we investigated the dynamics of glucose-dependent interaction and translocation of glucokinase and GRP in primary hepatocytes using fluorescence resonance energy transfer, selective photoconversion and fluorescence recovery after photobleaching. The formation of the glucokinase:GRP complex in the nucleus of primary hepatocytes at 5 mmol/l glucose was significantly reduced after a 2 h incubation at 20 mmol/l glucose. The GRP was predominantly localized in the nucleus, but a mobile fraction moved between the nucleus and the cytoplasm. The glucose concentration only marginally affected GRP shuttling. In contrast, the nuclear export rate of glucokinase was significantly higher at 20 than at 5 mmol/l glucose. Thus, glucose was proven to be the driving-force for nuclear export of glucokinase in hepatocytes. Using the FLII12Pglu-700μ-δ6 glucose nanosensor it could be shown that in hepatocytes the kinetics of nuclear glucose influx, metabolism or efflux were significantly faster compared to insulin-secreting cells. The rapid equilibration kinetics of glucose flux into the nucleus facilitates dissociation of the glucokinase:GRP complex and also nuclear glucose metabolism by free glucokinase enzyme. In conclusion, we could show that a rise of glucose in the nucleus of hepatocytes releases active glucokinase from the glucokinase:GRP complex and promotes the subsequent nuclear export of glucokinase. 相似文献
8.
Activation and translocation of glucokinase in rat primary hepatocytes monitored by high content image analysis 总被引:1,自引:0,他引:1
In the liver, glucokinase (GK) regulatory protein (GKRP) negatively modulates the metabolic enzyme GK by locking it in an inactive state in the nucleus. Here, the authors established a high content screening assay in the 384-well microplate format to measure the nucleus-to-cytoplasm translocation of GK by reagents that destabilize the interaction between GK and GKRP. As a cellular model system, primary rat hepatocytes endogenously expressing both GK and GKRP at physiological levels were used. The GK translocation assay was robust, displayed limited day-to-day variability, and delivered good Z' statistics. The increase of the glucose concentration in the extracellular medium from a low glucose situation (2.8 mM) to beyond its physiological set point value of 5 mM was found to drive GK from the nucleus into the cytoplasm. Likewise, both fructose (converted intracellularly into fructose-1-phosphate) and a known allosteric GK activator were found to induce the export of GK from the nucleus and to synergistically enhance the effects of medium or high glucose concentrations with respect to GK translocation. Transfer of the high content screening format to a semiautomated medium throughput screening platform enabled the profiling of large compound numbers with respect to allosteric activation of GK. 相似文献
9.
Grimsby J Coffey JW Dvorozniak MT Magram J Li G Matschinsky FM Shiota C Kaur S Magnuson MA Grippo JF 《The Journal of biological chemistry》2000,275(11):7826-7831
The glucokinase regulatory protein (GKRP) inhibits glucokinase competitively with respect to glucose by forming a protein-protein complex with this enzyme. The physiological role of GKRP in controlling hepatic glucokinase activity was addressed using gene targeting to disrupt GKRP gene expression. Heterozygote and homozygote knockout mice have a substantial decrease in hepatic glucokinase expression and enzymatic activity as measured at saturating glucose concentrations when compared with wild-type mice, with no change in basal blood glucose levels. Interestingly, when assayed under conditions to promote the association between glucokinase and GKRP, liver glucokinase activity in wild-type and null mice displayed comparable glucose phosphorylation capacities at physiological glucose concentrations (5 mM). Thus, despite reduced hepatic glucokinase expression levels in the null mice, glucokinase activity in the liver homogenates was maintained at nearly normal levels due to the absence of the inhibitory effects of GKRP. However, following a glucose tolerance test, the homozygote knockout mice show impaired glucose clearance, indicating that they cannot recruit sufficient glucokinase due to the absence of a nuclear reserve. These data suggest both a regulatory and a stabilizing role for GKRP in maintaining adequate glucokinase in the liver. Furthermore, this study provides evidence for the important role GKRP plays in acutely regulating of hepatic glucose metabolism. 相似文献
10.
Angel Gutierrez-Nogués Carmen-María García-Herrero Josep Oriola Olivier Vincent María-Angeles Navas 《生物化学与生物物理学报:疾病的分子基础》2018,1864(7):2385-2394
Glucokinase (GCK) plays a key role in glucose homeostasis. Heterozygous inactivating mutations in the GCK gene cause the familial, mild fasting hyperglycaemia named MODY2. Besides its particular kinetic characteristics, glucokinase is regulated by subcellular compartmentation in hepatocytes. Glucokinase regulatory protein (GKRP) binds to GCK, leading to enzyme inhibition and import into the nucleus at fasting. When glucose concentration increases, GCK-GKRP dissociates and GCK is exported to the cytosol due to a nuclear export signal (NES). With the aim to characterize the GCK-NES, we have functionally analysed nine MODY2 mutations located within the NES sequence.Recombinant GCK mutants showed reduced catalytic activity and, in most cases, protein instability. Most of the mutants interact normally with GKRP, although mutations L306R and L309P impair GCK nuclear import in cotransfected cells. We demonstrated that GCK-NES function depends on exportin 1. We further showed that none of the mutations fully inactivate the NES, with the exception of mutation L304P, which likely destabilizes its α-helicoidal structure. Finally, we found that residue Glu300 negatively modulates the NES activity, whereas other residues have the opposite effect, thus suggesting that some of the NES spacer residues contribute to the low affinity of the NES for exportin 1, which is required for its proper functioning.In conclusion, our results have provided functional and structural insights regarding the GCK-NES and contributed to a better knowledge of the molecular mechanisms involved in the nucleo-cytoplasmic shuttling of glucokinase. Impairment of this regulatory mechanism by some MODY2 mutations might contribute to the hyperglycaemia in the patients. 相似文献
11.
The association of glucokinase with liver mitochondria has been reported [Danial et al. (2003) BAD and glucokinase reside in a mitochondrial complex that integrates glycolysis and apoptosis. Nature 424, 952-956]. We confirmed association of glucokinase immunoreactivity with rat liver mitochondria using Percoll gradient centrifugation and demonstrated its association with the 68 kDa regulatory protein (GKRP) but not with the binding protein phosphofructokinase-2/fructose bisphosphatase-2. Substrates and glucagon induced adaptive changes in the mitochondrial glucokinase/GKRP ratio suggesting a regulatory role for GKRP. Combined with previous observations that GKRP overexpression partially inhibits glycolysis [de la Iglesia et al. (2000) The role of the regulatory protein of glucokinase in the glucose sensory mechanism of the hepatocyte. J. Biol. Chem. 275, 10597-10603] these findings suggest that there may be distinct glycolytic pools of glucokinase. 相似文献
12.
Glucokinase regulatory protein is essential for the proper subcellular localisation of liver glucokinase. 总被引:3,自引:0,他引:3
N de la Iglesia M Veiga-da-Cunha E Van Schaftingen J J Guinovart J C Ferrer 《FEBS letters》1999,456(2):332-338
Glucokinase (GK), a key enzyme in the glucose homeostatic responses of the liver, changes its intracellular localisation depending on the metabolic status of the cell. Rat liver GK and Xenopus laevis GK, fused to the green fluorescent protein (GFP), concentrated in the nucleus of cultured rat hepatocytes at low glucose and translocated to the cytoplasm at high glucose. Three mutant forms of Xenopus GK with reduced affinity for GK regulatory protein (GKRP) did not concentrate in the hepatocyte nuclei, even at low glucose. In COS-1 and HeLa cells, a blue fluorescent protein (BFP)-tagged version of rat liver GK was only able to accumulate in the nucleus when it was co-expressed with GKRP-GFP. At low glucose, both proteins concentrated in the nuclear compartment and at high glucose, BFP-GK translocated to the cytosol while GKRP-GFP remained in the nucleus. These findings indicate that the presence of and binding to GKRP are necessary and sufficient for the proper intracellular localisation of GK and directly involve GKRP in the control of the GK subcellular distribution. 相似文献
13.
Lentiviral genomic RNAs are encapsidated by the viral Gag protein during virion assembly. The intracellular location of the initial Gag-RNA interaction is unknown. We previously observed feline immunodeficiency virus (FIV) Gag accumulating at the nuclear envelope during live-cell imaging, which suggested that trafficking of human immunodeficiency virus type 1 (HIV-1) and FIV Gag may differ. Here we analyzed the nucleocytoplasmic transport properties of both Gag proteins. We discovered that inhibition of the CRM1 nuclear export pathway with leptomycin B causes FIV Gag but not HIV-1 Gag to accumulate in the nucleus. Virtually all FIV Gag rapidly became intranuclear when the CRM1 export pathway was blocked, implying that most if not all FIV Gag normally undergoes nuclear cycling. In FIV-infected feline cells, some intranuclear Gag was detected in the steady state without leptomycin B treatment. When expressed individually, the FIV matrix (MA), capsid (CA), and nucleocapsid-p2 (NC-p2) domains were not capable of mediating leptomycin B-sensitive nuclear export of a fluorescent protein. In contrast, CA-NC-p2 did mediate nuclear export, with MA being dispensable. We conclude that HIV-1 and FIV Gag differ strikingly in a key intracellular trafficking property. FIV Gag is a nuclear shuttling protein that utilizes the CRM1 nuclear export pathway, while HIV-1 Gag is excluded from the nucleus. These findings expand the spectrum of lentiviral Gag behaviors and raise the possibility that FIV genome encapsidation may initiate in the nucleus. 相似文献
14.
Joon Ha Park Choong Hyun Lee In Hye Kim Ji Hyeon Ahn Jeong-Hwi Cho Bing Chun Yan Jae-Chul Lee Tae Hun Lee Jeong Yeol Seo Jun Hwi Cho Moo-Ho Won Il-Jun Kang 《Neurochemical research》2013,38(12):2640-2649
Glucose is a main energy source for normal brain functions. Glucokinase (GK) plays an important role in glucose metabolism as a glucose sensor, and GK activity is modulated by glucokinase regulatory protein (GKRP). In this study, we examined the changes of GK and GKRP immunoreactivities in the gerbil hippocampus after 5 min of transient global cerebral ischemia. In the sham-operated-group, GK and GKRP immunoreactivities were easily detected in the pyramidal neurons of the stratum pyramidale of the hippocampus. GK and GKRP immunoreactivities in the pyramidal neurons were distinctively decreased in the hippocampal CA1 region (CA), not CA2/3, 3 days after ischemia–reperfusion (I–R). Five days after I–R, GK and GKRP immunoreactivities were hardly detected in the CA1, not CA2/3, pyramidal neurons; however, at this point in time, GK and GKRP immunoreactivities were newly expressed in astrocytes, not microglia, in the ischemic CA1. In brief, GK and GKRP immunoreactivities are changed in pyramidal neurons and newly expressed in astrocytes in the ischemic CA1 after transient cerebral ischemia. These indicate that changes of GK and GKRP expression may be related to the ischemia-induced neuronal damage/death. 相似文献
15.
16.
Upf3p, which is required for nonsense-mediated mRNA decay (NMD) in yeast, is primarily cytoplasmic but accumulates inside the nucleus when UPF3 is overexpressed or when upf3 mutations prevent nuclear export. Upf3p physically interacts with Srp1p (importin-alpha). Upf3p fails to be imported into the nucleus in a temperature-sensitive srp1-31 strain, indicating that nuclear import is mediated by the importin-alpha/beta heterodimer. Nuclear export of Upf3p is mediated by a leucine-rich nuclear export sequence (NES-A), but export is not dependent on the Crm1p exportin. Mutations identified in NES-A prevent nuclear export and confer an Nmd(-) phenotype. The addition of a functional NES element to an export-defective upf(-) allele restores export and partially restores an Nmd(+) phenotype. Our findings support a model in which the movement of Upf3p between the nucleus and the cytoplasm is required for a fully functional NMD pathway. We also found that overexpression of Upf2p suppresses the Nmd(-) phenotype in mutant strains carrying nes-A alleles but has no effect on the localization of Upf3p. To explain these results, we suggest that the mutations in NES-A that impair nuclear export cause additional defects in the function of Upf3p that are not rectified by restoration of export alone. 相似文献
17.
Bourbonais FJ Chen J Huang C Zhang Y Pfefferkorn JA Landro JA 《The Biochemical journal》2012,441(3):881-887
GK (glucokinase) is an enzyme central to glucose metabolism that displays positive co-operativity to substrate glucose. Small-molecule GKAs (GK activators) modulate GK catalytic activity and glucose affinity and are currently being pursued as a treatment for Type 2 diabetes. GK progress curves monitoring product formation are linear up to 1 mM glucose, but biphasic at 5 mM, with the transition from the lower initial velocity to the higher steady-state velocity being described by the rate constant kact. In the presence of a liver-specific GKA (compound A), progress curves at 1 mM glucose are similar to those at 5 mM, reflecting activation of GK by compound A. We show that GKRP (GK regulatory protein) is a slow tight-binding inhibitor of GK. Analysis of progress curves indicate that this inhibition is time dependent, with apparent initial and final Ki values being 113 and 12.8 nM respectively. When GK is pre-incubated with glucose and compound A, the inhibition observed by GKRP is time dependent, but independent of GKRP concentration, reflecting the GKA-controlled transition between closed and open GK conformations. These data are supported by cell-based imaging data from primary rat hepatocytes. This work characterizes the modulation of GK by a novel GKA that may enable the design of new and improved GKAs. 相似文献
18.
Tong EH Guo JJ Huang AL Liu H Hu CD Chung SS Ko BC 《The Journal of biological chemistry》2006,281(33):23870-23879
The osmotic response element-binding protein (OREBP), also known as tonicity enhancer-binding protein (TonEBP) or NFAT5, regulates the hypertonicity-induced expression of a battery of genes crucial for the adaptation of mammalian cells to extracellular hypertonic stress. The activity of OREBP/TonEBP is regulated at multiple levels, including nucleocytoplasmic trafficking. OREBP/TonEBP protein can be detected in both the cytoplasm and nucleus under isotonic conditions, although it accumulates exclusively in the nucleus or cytoplasm when subjected to hypertonic or hypotonic challenges, respectively. Using immunocytochemistry and green fluorescent protein fusions, the protein domains that determine its subcellular localization were identified and characterized. We found that OREBP/TonEBP nuclear import is regulated by a nuclear localization signal. However, under isotonic conditions, nuclear export of OREBP/TonEBP is mediated by a CRM1-dependent, leucine-rich canonical nuclear export sequence (NES) located in the N terminus. Disruption of NES by site-directed mutagenesis yielded a mutant OREBP/TonEBP protein that accumulated in the nucleus under isotonic conditions but remained a target for hypotonicity-induced nuclear export. More importantly, a putative auxiliary export domain distal to the NES was identified. Disruption of the auxiliary export domain alone is sufficient to abolish the nuclear export of OREBP/TonEBP induced by hypotonicity. By using bimolecular fluorescence complementation assay, we showed that CRM1 interacts with OREBP/TonEBP, but not with a mutant protein deficient in NES. Our findings provide insight into how nucleocytoplasmic trafficking of OREBP/TonEBP is regulated by changes in extracellular tonicity. 相似文献
19.
Identification of fructose 6-phosphate- and fructose 1-phosphate-binding residues in the regulatory protein of glucokinase. 总被引:2,自引:0,他引:2
Maria Veiga-da-Cunha Emile Van Schaftingen 《The Journal of biological chemistry》2002,277(10):8466-8473
Glucokinase is inhibited in the liver by a regulatory protein (GKRP) whose effects are increased by Fru-6-P and suppressed by Fru-1-P. To identify the binding site of these phosphate esters, we took advantage of the homology of GKRP to the isomerase domain of GlmS (glucosamine-6-phosphate synthase) and created 12 different mutants of rat GKRP. Mutations of three residues predicted to bind to Fru-6-P resulted in proteins that were approximately 5-fold (S110A) and 50-fold (S179A and K514A) less potent as inhibitors of glucokinase and had an at least 100-fold reduced affinity for the effectors. Mutation of another residue of the putative binding site (T109A) resulted in a 10-fold decrease in the inhibitory power and an inversion of the effect of sorbitol-6-P, a Fru-6-P analog. The replacement of Gly(107), a residue close to the binding site, by cysteine (as in GlmS and Xenopus GKRP) resulted in a protein that had 20 times more affinity for Fru-6-P and 30 times less affinity for Fru-1-P. These results are consistent with GKRP having one single binding site for phosphate esters. They also show that a missense mutation of GKRP can lead to a gain of function. 相似文献
20.
Yagisawa H 《Journal of cellular biochemistry》2006,97(2):233-243
Phosphoinositides (PIs) and proteins involved in the PI signaling pathway are distributed in the nucleus as well as at the plasma membrane and in the cytoplasm, although their nuclear localization mechanisms have not been clarified in detail. Generally, proteins that shuttle between the cytoplasm and nucleus contain nuclear localization signal (NLS) and nuclear export signal (NES) sequences for nuclear import and export, respectively. They bind to specific carrier proteins of the importin/exportin family and are transported to and from the nucleus. Thus there is a steady state shuttling of the cargo molecules to and from the nucleus, and the shift in equilibrium determines their nuclear or cytoplasmic localization. Our previous studies have shown that phospholipase C (PLC)-delta1, regarded as having cytoplasmic- or plasma membrane-bound localization, accumulates in the nucleus when its NES sequence is disrupted. In addition, a cluster of positively charged residues on the surface of the catalytic barrel is important for nuclear import. In quiescent cells, the shuttling equilibrium seems to be shifted to the nuclear export of PLCdelta1. In this review, recent findings regarding the molecular machineries and mechanisms of the nucleocytoplasmic shuttling of PLCdelta1 will be discussed. It is important to know when and how they are regulated. A shift in the equilibrium in a certain stage of the cell cycle or by external stimuli is possible and resulting changes in the intra-nuclear environments (or architectures) may alter proliferation and differentiation patterns. Evidences support the idea that an increase in the levels of intracellular Ca2+ shifts the equilibrium to the nuclear import of PLCdelta1. A myriad of external stimuli have also been reported to change the nuclear PI metabolism following accelerated accumulation in the nucleus of other phospholipases such as phospholipase A2 and phospholipase D in addition to PLC isoforms such as PLCbeta1 and PLCgamma1. The consequence of the nuclear accumulation of PLC is also discussed. 相似文献