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1.
We examined the mechanisms involved in protein kinase C (PKC)-dependent down-regulation of dopamine transporter (DAT) activity and cell surface expression by treating heterologously expressing cells with the clathrin-mediated endocytosis inhibitor concanavalin A (Con A) or the cholesterol depleter/membrane raft disrupter methyl-β-cyclodextrin (MβC) prior to treatment with the PKC activator phorbol 12-myristate, 13-acetate (PMA). Con A blocked PMA-induced surface reductions of DAT but only partially inhibited down-regulation, while MβC partially blocked down-regulation but did not inhibit loss of cell surface DAT, demonstrating that PKC-induced DAT down-regulation occurs by a combination of trafficking and non-trafficking processes. Using density-gradient centrifugation, we found that DATs are distributed approximately equally between Triton-insoluble, cholesterol-rich membrane rafts and Triton-soluble non-raft membranes. DATs in both populations are present at the cell surface and are active for dopamine and cocaine binding. PMA-induced loss of cell surface DAT occurred only from non-raft populations, demonstrating that non-raft DATs are regulated by trafficking events and indicating the likelihood that the cholesterol-dependent non-trafficking regulatory mechanism occurs in rafts. PMA did not affect the DAT raft-non-raft distribution but stimulated the phosphorylation of DAT to a substantially greater level in rafts than non-rafts. These findings reveal a previously unknown role for cholesterol in DAT function and demonstrate the presence of distinct subcellular DAT populations that possess multiple regulatory differences that may impact dopaminergic neurotransmission.  相似文献   

2.
3.
Abstract: Neurotransmission at excitatory glutamatergic synapses is terminated by the reuptake of the neurotransmitter by high-affinity transporters, which keep the extracellular glutamate concentration below excitotoxic levels. The amino acid sequence of the recently isolated and cloned brain-specific glutamate/aspartate transporter (GLAST-1) of the rat reveals three consensus sequences of putative phosphorylation sites for protein kinase C (PKC). The PKC activator phorbol 12-myristate 13-acetate (PMA) decreased glutamate transport activity in Xenopus oocytes and human embryonic kidney cells (HEK293) expressing the cloned GLAST-1 cDNA, within 20 min, to 25% of the initial transport activity. This down-regulation was blocked by the PKC inhibitor staurosporine. GLAST-1 transport activity remains unimpaired by phorbol 12-monomyristate. Removal of all putative PKC sites of wild-type GLAST-1 by site-directed mutagenesis did not abolish inhibition of glutamate transport. [32P]Phosphate-labeled wild-type and mutant transport proteins devoid of all predicted PKC sites were detected by immunoprecipitation after stimulation with PMA. Immunoprecipitation of [35S]methionine-labeled transporter molecules indicates a similar stability of phosphorylated and nonphosphorylated GLAST-1 protein. Immunofluorescence staining did not differentiate surface staining of HEK293 cells expressing GLAST-1 with and without PMA treatment. These data suggest that the neurotransmitter transporter activity of GLAST-1 is inhibited by phosphorylation at a non-PKC consensus site.  相似文献   

4.
Doolen S  Zahniser NR 《FEBS letters》2002,516(1-3):187-190
The hypothesis that specific protein kinase C (PKC) isoforms regulate dopamine transporter (DAT) function was tested in Xenopus laevis oocytes expressing human (h)DAT. Activation of conventional PKCs (cPKCs) and novel PKCs (nPKCs) using 10 nM phorbol 12-myristate 13-acetate (PMA) significantly inhibited DAT-associated transport currents. This effect was reversed by isoform-non-selective PKC inhibitors, selective inhibitors of cPKCs and deltaPKC, and by Ca2+ chelation. By contrast, the epsilonPKC translocation inhibitor peptide had no effect on PMA-induced inhibition of hDAT transport-associated currents. Thus, the primary mechanism by which PMA regulates hDAT expressed in oocytes appears to be by activating cPKC(s).  相似文献   

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6.
The human cationic amino acid transporter hCAT-1 is almost ubiquitously expressed and probably the most important entity for supplying cells with extracellular arginine, lysine, and ornithine. We have previously shown that hCAT-1-mediated transport is decreased after protein kinase C (PKC) activation by phorbol 12-myristate 13-acetate (PMA) (Gr?f, P., Forstermann, U., and Closs, E. I. (2001) Br. J. Pharmacol. 132, 1193-1200). In the present study, we examined the mechanism of this down-regulation. In both Xenopus laevis oocytes and U373MG glioblastoma cells, PMA treatment promoted the internalization of hCAT-1 (fused to the enhanced green fluorescence protein (EGFP)) as visualized by fluorescence microscopy. Biotinylation of cell surface proteins and subsequent Western blot analyses confirmed that the cell surface expression of hCAT-1.EGFP was significantly reduced upon PMA treatment. Pretreatment with the PKC inhibitor bisindolylmaleimide I prevented the reduction by PMA of both hCAT-1.EGFP-induced arginine transport and the internalization of the transporter. Similar results were obtained with hCAT-1 expressed endogenously in DLD-1 colon carcinoma cells. Inhibition of protein synthesis did not augment the PMA effect. In addition, the PMA effect was reverted in washout experiments without changing the hCAT-1 protein expression, suggesting that the PMA effect is reversible in these cells. PKC did not phosphorylate hCAT-1 directly as evidenced by in vivo phosphorylation experiments and mutational analysis, indicating an indirect action of PKC on hCAT-1.  相似文献   

7.
Protein kinase C (PKC) regulation of l-ascorbic acid transport mediated by the Na+/ascorbic acid transporters, hSVCT1 and hSVCT2, expressed in COS-1 cells was studied using recombinant carboxyl-terminal V5 epitope-tagged forms of the transporters. The PKC activator phorbol 12-myristate 13-acetate (PMA) caused a time-dependent and concentration-dependent decrease (40-60%) in ascorbic acid transport activity. Effects of PMA were not observed with the inactive phorbol ester 4 alpha-phorbol and were reversed by treatment of the cells with the PKC-specific inhibitor Ro-31-8220. Kinetically, the reduction in hSVCT1 and hSVCT2 activity arose from a decrease in maximal velocity with no change in the apparent affinity. Western blot and confocal microscopy analyses indicated that the total pool of hSVCT1 or hSVCT2 proteins expressed in the transfected COS-1 cells remained unaffected by PMA treatment. For hSVCT1 the decrease in L-ascorbic acid correlated with a redistribution of the transporter from the cell surface to intracellular membranes. However, for hSVCT2 there was no apparent change in transporter distribution, suggesting that the PKC-dependent modulation of L-ascorbic acid transport mediated by hSVCT2 was the result of reduced catalytic transport efficiency.  相似文献   

8.
L-carnitine is absorbed in the intestinal tract via the carnitine transporter OCTN2 and the amino acid transporter ATB(0,+). Loss-of-function mutations in OCTN2 may be associated with inflammatory bowel disease (IBD), suggesting a role for carnitine in intestinal/colonic health. In contrast, ATB(0,+) is upregulated in bowel inflammation. Butyrate, a bacterial fermentation product, is beneficial for prevention/treatment of ulcerative colitis. Butyryl-L-carnitine (BC), a butyrate ester of carnitine, may have potential for treatment of gut inflammation, since BC would supply both butyrate and carnitine. We examined the transport of BC via ATB(0,+) to determine if this transporter could serve as a delivery system for BC. We also examined the transport of BC via OCTN2. Studies were done with cloned ATB(0,+) and OCTN2 in heterologous expression systems. BC inhibited ATB(0,+)-mediated glycine transport in mammalian cells (IC(50), 4.6 +/- 0.7 mM). In Xenopus laevis oocytes expressing human ATB(0,+), BC induced Na(+) -dependent inward currents under voltage-clamp conditions. The currents were saturable with a K(0.5) of 1.4 +/- 0.1 mM. Na(+) activation kinetics of BC-induced currents suggested involvement of two Na(+) per transport cycle. BC also inhibited OCTN2-mediated carnitine uptake (IC(50), 1.5 +/- 0.3 microM). Transport of BC via OCTN2 is electrogenic, as evidenced from BC-induced inward currents. These currents were Na(+) dependent and saturable (K(0.5), 0.40 +/- 0.02 microM). We conclude that ATB(0,+) is a low-affinity/high-capacity transporter for BC, whereas OCTN2 is a high-affinity/low-capacity transporter. ATB(0,+) may mediate intestinal absorption of BC when OCTN2 is defective.  相似文献   

9.
D-Serine, synthesized endogenously in the brain, is an important modulator of glutamatergic neurotransmission. Since colonic bacteria produce D-serine, we asked the question whether there are transport mechanisms in the colon that might make this exogenously produced D-serine available to the host. Here we identify for the first time an amino acid transporter in the intestine for high-affinity active transport of D-serine. This transporter, called ATB(0,+), is a Na(+)- and Cl(-)-coupled transporter for L-enantiomers of neutral and cationic amino acids. Here we demonstrate that ATB(0,+) is also capable of mediating the Na(+)- and Cl(-)-coupled transport of D-serine. The affinity of ATB(0,+) for L-serine and D-serine is similar, the K(t) value for the two enantiomers being approximately 150 microM. In addition to D-serine, ATB(0,+) transports D-alanine, D-methionine, D-leucine, and D-tryptophan. However, several other neutral and cationic amino acids that are transportable substrates for ATB(0,+) as L-enantiomers are not transported when presented as D-enantiomers. ATB(0,+) is expressed in the intestinal tract, interestingly not in the proximal intestine but in the distal intestine. Expression is most predominant in the colon where the transporter is localized to the luminal membrane of colonocytes, making this transporter uniquely suitable for absorption of bacteria-derived D-serine.  相似文献   

10.
Recent studies have highlighted the existence of discrete microdomains at the cell surface that are distinct from caveolae. The function of these microdomains remains unknown. However, recent evidence suggests that they may participate in a subset of transmembrane signaling events. In hematopoietic cells, these low density Triton-insoluble (LDTI) microdomains (also called caveolae-related domains) are dramatically enriched in signaling molecules, such as cell surface receptors (CD4 and CD55), Src family tyrosine kinases (Lyn, Lck, Hck, and Fyn), heterotrimeric G proteins, and gangliosides (GM1 and GM3). Human T lymphocytes have become a well established model system for studying the process of phorbol ester-induced down-regulation of CD4. Here, we present evidence that phorbol 12-myristate 13-acetate (PMA)-induced down-regulation of the cell surface pool of CD4 occurs within the LDTI microdomains of T cells. Localization of CD4 in LDTI microdomains was confirmed by immunoelectron microscopy. PMA-induced disruption of the CD4-Lck complex was rapid (within 5 min), and this disruption occurred within LDTI microdomains. Because PMA is an activator of protein kinase C (PKC), we next evaluated the possible roles of different PKC isoforms in this process. Our results indicate that PMA induced the rapid translocation of cytosolic PKCs to LDTI microdomains. We identified PKCalpha as the major isoform involved in this translocation event. Taken together, our results support the hypothesis that LDTI microdomains represent a functionally important plasma membrane compartment in T cells.  相似文献   

11.
The effect of phorbol 12-myristate 13-acetate on the phosphorylation of the ras p21 protein was studied by metabolically labeling cultured cells with [32P]orthophosphate and using a monoclonal antibody to immunoprecipitate the protein. Phorbol 12-myristate 13-acetate (100 nM) induced phosphorylation of cKi-ras p21 in a mouse adrenocortical cell line (Yl) expressing high levels of cKi-ras with exon 4B. Phosphorylation was detected at 10 min and was maximal at 2 h. The ras protein was not phosphorylated in response to phorbol 12-myristate 13-acetate in NIH 3T3 cells expressing activated cHa-ras or vHa-ras. In vitro, protein kinase C phosphorylated cKi-ras in a phosphatidylserine and diolein-dependent manner. Both in intact cells and in vitro the amino acid phosphorylated was serine. Analysis of p21 from NIH 3T3 cells expressing a variety of ras proteins indicated that phosphorylation occurs within a domain encoded by exon 4B of cKi-ras. Phosphorylation affected neither the binding nor the GTPase activity of the ras protein. We conclude that cKi-ras is a substrate for protein kinase C and that the site of phosphorylation is likely to be serine 181 encoded by exon 4B.  相似文献   

12.
The cAMP-response element-binding protein (CREB) is activated by phosphorylation on Ser-133 and plays a key role in the proliferative and survival responses of mature B cells to B cell receptor (BCR) signaling. The signal link between the BCR and CREB activation depends on a phorbol ester (phorbol 12-myristate 13-acetate)-sensitive protein kinase C (PKC) activity and not protein kinase A or calmodulin kinase; however, the identity and role of the PKC(s) activity has not been elucidated. We found the novel PKCdelta (nPKCdelta) activator bistratene A is sufficient to induce CREB phosphorylation in murine splenic B cells. The pharmacological inhibitor G?6976, which targets conventional PKCs and PKCmu, has no effect on CREB phosphorylation, whereas the nPKCdelta inhibitor rottlerin blocks CREB phosphorylation following BCR cross-linking. Bryostatin 1 selectively prevents nPKCdelta depletion by phorbol 12-myristate 13-acetate when coapplied, coincident with protection of BCR-induced CREB phosphorylation. Ectopic expression of a kinase-inactive nPKCdelta blocks BCR-induced CREB phosphorylation in A20 B cells. In addition, BCR-induced CREB phosphorylation is significantly diminished in nPKCdelta-deficient splenic B cells in comparison with wild type mice. Consistent with the essential role for Bruton's tyrosine kinase and phospholipase Cgamma2 in mediating PKC activation, Bruton's tyrosine kinase- and phospholipase Cgamma2-deficient B cells display defective CREB phosphorylation by the BCR. We also found that p90 RSK directly phosphorylates CREB on Ser-133 following BCR cross-linking and is positioned downstream of nPKCdelta. Taken together, these results suggest a model in which BCR engagement leads to the phosphorylation of CREB via a signaling pathway that requires nPKCdelta and p90 RSK in mature B cells.  相似文献   

13.
Two protein kinase C isoenzymes were partially purified from the nuclei of human neutrophils, and identified as beta and alpha subtypes. Treatment of neutrophils with phorbol 12-myristate 13-acetate (PMA) caused a 3.8-fold increase of nuclear beta PKC activity, while a minor increase of alpha PKC was observed. This selective activation of beta PKC could help to understand the molecular events involved in phorbol ester-induced cellular modifications.  相似文献   

14.
Angiotensin-converting enzyme (ACE) is an extensively glycosylated type I ectoprotein anchored in the plasma membrane by a hydrophobic transmembrane domain. In tissue culture as well as in vivo, the extracellular domain of ACE is released into the culture medium by a regulated proteolytic cleavage. To identify the cellular proteins that regulate ACE processing and cleavage-secretion, ACE-bound proteins were purified by affinity chromatography and characterized by microsequencing and Western blotting. One protein was identified as ribophorin and another as immunoglobulin-binding protein (BiP), a chaperone. Metabolic labeling and immunoprecipitation of ACE confirmed its interaction with BiP. Overexpression of BiP inhibited ACE secretion, an effect accentuated by the expression of an enzymatically inactive mutant BiP. This inhibition was caused by the retention of ACE precursors by BiP in the endoplasmic reticulum, as revealed by immunoprecipitation and immunofluorescence experiments. However, treatment with a phorbol ester, phorbol 12-myristate 13-acetate, enhanced ACE secretion even from cells overexpressing BiP. Western blot analysis of ACE-associated proteins with antibodies to protein kinase C (PKC) revealed the presence of its specific isozymes. Treatment with phorbol 12-myristate 13-acetate caused marked reduction in ACE association of selective PKC species. Thus, our studies have identified PKC and BiP as two proteins that directly interact with ACE and modulate its cell-surface expression and cleavage-secretion.  相似文献   

15.
Pertussis toxin (PTX) induces activation of l-arginine transport in pulmonary artery endothelial cells (PAEC). The effects of PTX on l-arginine transport appeared after 6 h of treatment and reached maximal values after treatment for 12 h. PTX-induced changes in l-arginine transport were not accompanied by changes in expression of cationic amino acid transporter (CAT)-1 protein, the main l-arginine transporter in PAEC. Unlike holotoxin, the beta-oligomer-binding subunit of PTX did not affect l-arginine transport in PAEC, suggesting that Galpha(i) ribosylation is an important step in the activation of l-arginine transport by PTX. An activator of adenylate cyclase, forskolin, and an activator of protein kinase A (PKA), Sp-cAMPS, did not affect l-arginine transport in PAEC. In addition, inhibitors of PKA or adenylate cyclase did not change the activating effect of PTX on l-arginine uptake. Long-term treatment with PTX (18 h) induced a 40% decrease in protein kinase C (PKC)-alpha but did not affect the activities of PKC-epsilon and PKC-zeta in PAEC. An activator of PKC-alpha, phorbol 12-myristate 13-acetate, abrogated the activation of l-arginine transport in PAEC treated with PTX. Incubation of PTX-treated PAEC with phorbol 12-myristate 13-acetate in combination with an inhibitor of PKC-alpha (Go 6976) restored the activating effects of PTX on l-arginine uptake, suggesting PTX-induced activation of l-arginine transport is mediated through downregulation of PKC-alpha. Measurements of nitric oxide (NO) production by PAEC revealed that long-term treatment with PTX induced twofold increases in the amount of NO in PAEC. PTX also increased l-[(3)H]citrulline production from extracellular l-[(3)H]arginine without affecting endothelial NO synthase activity. These results demonstrate that PTX increased NO production through activation of l-arginine transport in PAEC.  相似文献   

16.
ATB(0,+) (SLC6A14) is a Na(+)/Cl(-)-coupled arginine transporter expressed at low levels in normal colon. Arginine is an essential amino acid for tumor cells. Arginine is also the substrate for nitric oxide synthases (NOSs). Since arginine and arginine-derived nitric oxide (NO) play a critical role in cancer, we examined the expression of ATB(0,+) in colorectal cancer. Paired normal and cancer tissues from colectomy specimens of 10 patients with colorectal cancer and from the liver tissue of one patient with hepatic metastasis from a colonic primary were used for the analysis of the levels of ATB(0,+) mRNA, inducible NOS (iNOS) mRNA and the corresponding proteins. Tissues samples from the colon, liver, and lymph nodes of an additional patient with metastatic colon cancer were analyzed for ATB(0,+) protein alone. We also examined the levels of nitrotyrosylated proteins. The ATB(0,+) mRNA increased 22.9+/-3.0-fold in colorectal cancer compared to normal tissue and the increase was evident in each of the 10 cases examined. iNOS mRNA increased 5.2+/-1.1-fold in cancer specimens. The changes in mRNA levels were associated with an increase in ATB(0,+), iNOS, and nitrotyrosylated proteins. The increased expression of ATB(0,+) and iNOS was also demonstrated in liver and lymph node specimens with metastases from colonic primaries. This study strongly suggests that the upregulation of ATB(0,+) may have a pathogenic role in colorectal cancer. Since ATB(0,+) is a versatile transporter not only for arginine but also for several drugs including NOS inhibitors, these findings have significant clinical and therapeutic relevance.  相似文献   

17.
We provide evidence here that b(0,+) amino acid transporter (b(0, +)AT) interacts with 4F2 heavy chain (4F2hc) as well as with the protein related to b(0,+) amino acid transporter (rBAT) to constitute functionally competent b(0,+)-like amino acid transport systems. This evidence has been obtained by co-expression of b(0, +)AT and 4F2hc or b(0,+)AT and rBAT in human retinal pigment epithelial cells and in COS-1 cells. The ability to interact with 4F2hc and rBAT is demonstrable with mouse b(0,+)AT as well as with human b(0,+)AT. Even though both the 4F2hc x b(0,+)AT complex and the rBAT x b(0,+)AT complex exhibit substrate specificity that is characteristic of system b(0,+), these two complexes differ significantly in substrate affinity. The 4F2hc x b(0,+)AT complex has higher substrate affinity than the rBAT x b(0,+)AT complex. In situ hybridization studies demonstrate that the regional distribution pattern of mRNA in the kidney is identical for b(0,+)AT and 4F2hc. The pattern of rBAT mRNA expression is different from that of b(0,+)AT mRNA and 4F2hc mRNA, but there are regions in the kidney where b(0,+)AT mRNA expression overlaps with rBAT mRNA expression as well as with 4F2hc mRNA expression.  相似文献   

18.
We examined which isoforms of protein kinase C (PKC) may be involved in the regulation of cationic amino acid transporter-1 (CAT-1) transport activity in cultured pulmonary artery endothelial cells (PAEC). An activator of classical and novel isoforms of PKC, phorbol 12-myristate-13-acetate (PMA; 100 nM), inhibited CAT-1-mediated l-arginine transport in PAEC after a 1-h treatment and activated l-arginine uptake after an 18-h treatment of cells. These changes in l-arginine transport were not related to the changes in the expression of the CAT-1 transporter. The inhibitory effect of PMA on l-arginine transport was accompanied by a translocation of PKCalpha (a classical PKC isoform) from the cytosol to the membrane fraction, whereas the activating effect of PMA on l-arginine transport was accompanied by full depletion of the expression of PKCalpha in PAEC. A selective activator of Ca(2+)-dependent classical isoforms of PKC, thymeleatoxin (Thy; 100 nM; 1-h and 18-h treatments), induced the same changes in l-arginine uptake and PKCalpha translocation and depletion as PMA. The effects of PMA and Thy on l-arginine transport in PAEC were attenuated by a selective inhibitor of classical PKC isoforms Go 6976 (1 micro M). Phosphatidylinositol-3,4,5-triphosphate-dipalmitoyl (PIP; 5 micro M), which activates novel PKC isoforms, did not affect l-arginine transport in PAEC after 1-h and 18-h treatment of cells. PIP (5 micro M; 1 h) induced the translocation of PKCepsilon (a novel PKC isoform) from the cytosolic to the particulate fraction and did not affect the translocation of PKCalpha. These results demonstrate that classical isoforms of PKC are involved in the regulation of CAT-1 transport activity in PAEC. We suggest that translocation of PKCalpha to the plasma membrane induces phosphorylation of the CAT-1 transporter, which leads to inhibition of its transport activity in PAEC. In contrast, depletion of PKCalpha after long-term treatment with PMA or Thy promotes dephosphorylation of the CAT-1 transporter and activation of its activity.  相似文献   

19.
ATB(0,+) [SLC6A14 (solute carrier family 6 member 14)] is an Na(+)/Cl(-)-coupled amino acid transporter whose expression is upregulated in cancer. 1-Methyltryptophan is an inducer of immune surveillance against tumour cells through its ability to inhibit indoleamine dioxygenase. In the present study, we investigated the role of ATB(0,+) in the uptake of 1-methyltryptophan as a potential mechanism for entry of this putative anticancer drug into tumour cells. These studies show that 1-methyltryptophan is a transportable substrate for ATB(0,+). The transport process is Na(+)/Cl(-)-dependent with an Na(+)/Cl(-)/1-methyltryptophan stoichiometry of 2:1:1. Evaluation of other derivatives of tryptophan has led to identification of alpha-methyltryptophan as a blocker, not a transportable substrate, for ATB(0,+). ATB(0,+) can transport 18 of the 20 proteinogenic amino acids. alpha-Methyltryptophan blocks the transport function of ATB(0,+) with an IC(50) value of approximately 250 muM under conditions simulating normal plasma concentrations of all these 18 amino acids. These results suggest that alpha-methyltryptophan may induce amino acid deprivation in cells which depend on the transporter for their amino acid nutrition. Screening of several mammary epithelial cell lines shows that ATB(0,+) is expressed robustly in some cancer cell lines, but not in all; in contrast, non-malignant cell lines do not express the transporter. Treatment of ATB(0,+)-positive tumour cells with alpha-methyltryptophan leads to suppression of their colony-forming ability, whereas ATB(0,+)-negative cell lines are not affected. The blockade of ATB(0,+) in these cells with alpha-methyltryptophan is associated with cell cycle arrest. These studies reveal the potential of ATB(0,+) as a drug target for cancer chemotherapy.  相似文献   

20.
Abstract: Treatment of human embryonic kidney cells (HEK 293 cells) expressing the mouse glycine transporter 1 (GLYT1b) with the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) decreased specific [3H]glycine uptake. This down-regulation resulted from a reduction of the maximal transport rate and was blocked by the PKC inhibitors 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7) and staurosporine. The inhibitory effect of PMA treatment was also observed after removing all five predicted phosphorylation sites for PKC in GLYT1b by site-directed mutagenesis. These data indicate that glycine transport by GLYT1b is modulated by PKC activation; however, this regulation may involve indirect phosphorylation mechanisms.  相似文献   

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