Phosphofructokinase Muscle-Specific Isoform Requires Caveolin-3 Expression for Plasma Membrane Recruitment and Caveolar Targeting
2003; Elsevier BV; Volume: 163; Issue: 6 Linguagem: Inglês
10.1016/s0002-9440(10)63616-4
ISSN1525-2191
AutoresFederica Sotgia, Gloria Bonuccelli, Carlo Minetti, Scott E. Woodman, Franco Capozza, Robert G. Kemp, Philipp E. Scherer, Michael P. Lisanti,
Tópico(s)Erythrocyte Function and Pathophysiology
ResumoPrevious co-immunoprecipitation studies have shown that endogenous PFK-M (phosphofructokinase, muscle-specific isoform) associates with caveolin (Cav)-3 under certain metabolic conditions. However, it remains unknown whether Cav-3 expression is required for the plasma membrane recruitment and caveolar targeting of PFK-M. Here, we demonstrate that recombinant expression of Cav-3 dramatically affects the subcellular localization of PFK-M, by targeting PFK-M to the plasma membrane, and by trans-locating PFK-M to caveolae-enriched membrane domains. In addition, we show that the membrane recruitment and caveolar targeting of PFK-M appears to be strictly dependent on the concentration of extracellular glucose. Interestingly, recombinant expression of PFK-M with three Cav-3 mutants [ΔTFT (63 to 65), P104L, and R26Q], which harbor the same mutations as seen in the human patients with Cav-3-related muscle diseases, causes a substantial reduction in PFK-M expression levels, and impedes the membrane recruitment of PFK-M. Analysis of skeletal muscle tissue samples from Cav-3(−/−) mice directly demonstrates that Cav-3 expression regulates the phenotypic behavior of PFK-M. More specifically, in Cav-3-null mice, PFK-M is no longer targeted to the plasma membrane, and is excluded from caveolar membrane domains. As such, our current results may be important in understanding the pathogenesis of Cav-3-related muscle diseases, such as limb-girdle muscular dystrophy-1C, distal myopathy, and rippling muscle disease, that are caused by mutations within the human Cav-3 gene. Previous co-immunoprecipitation studies have shown that endogenous PFK-M (phosphofructokinase, muscle-specific isoform) associates with caveolin (Cav)-3 under certain metabolic conditions. However, it remains unknown whether Cav-3 expression is required for the plasma membrane recruitment and caveolar targeting of PFK-M. Here, we demonstrate that recombinant expression of Cav-3 dramatically affects the subcellular localization of PFK-M, by targeting PFK-M to the plasma membrane, and by trans-locating PFK-M to caveolae-enriched membrane domains. In addition, we show that the membrane recruitment and caveolar targeting of PFK-M appears to be strictly dependent on the concentration of extracellular glucose. Interestingly, recombinant expression of PFK-M with three Cav-3 mutants [ΔTFT (63 to 65), P104L, and R26Q], which harbor the same mutations as seen in the human patients with Cav-3-related muscle diseases, causes a substantial reduction in PFK-M expression levels, and impedes the membrane recruitment of PFK-M. Analysis of skeletal muscle tissue samples from Cav-3(−/−) mice directly demonstrates that Cav-3 expression regulates the phenotypic behavior of PFK-M. More specifically, in Cav-3-null mice, PFK-M is no longer targeted to the plasma membrane, and is excluded from caveolar membrane domains. As such, our current results may be important in understanding the pathogenesis of Cav-3-related muscle diseases, such as limb-girdle muscular dystrophy-1C, distal myopathy, and rippling muscle disease, that are caused by mutations within the human Cav-3 gene. Phosphofructokinase (PFK) is an enzyme of central importance in the regulation of carbohydrate metabolism. It catalyzes the rate-limiting and irreversible reaction in glycolysis, the conversion of fructose-6-phosphate to fructose-1,6-bisphosphate. Because PFK plays such a key role in the control of the glycolytic pathway, its activity appears to be tightly regulated and modulated by several allosteric effectors.1Kemp RG Foe LG Allosteric regulatory properties of muscle phosphofructokinase.Mol Cell Biochem. 1983; 57: 147-154Crossref PubMed Scopus (116) Google Scholar It is inhibited by high concentrations of ATP and citrate, and it is stimulated by AMP, Pi, fructose-2,6-bisphosphate, and one of the products of its own reaction, fructose-1,6-bisphosphate. Therefore, whenever the cell has high concentrations of ATP, or other sources of energy are available, such as citrate or long-chain fatty acids, PFK activity is inhibited and glycolysis is blocked. On the other hand, when the cell is in energy demand, PFK activity is stimulated. Thus, it is believed that, in resting cells, the enzyme is functionally inactive.Three different PFK isoforms have been identified in mammals, termed 1) PFK-A or PFK-M (muscle-type), 2) PFK-B (PFK-L in human, liver-type), and 3) PFK-C or PFK-P (platelet-type)—each encoded by a different gene.2Tsai MY Kemp RG Hybridization of rabbit muscle and liver phosphofructokinases.Arch Biochem Biophys. 1972; 150: 407-411Crossref PubMed Scopus (15) Google Scholar, 3Dunaway GA Kasten TP Sebo T Trapp R Analysis of the phosphofructokinase subunits and isoenzymes in human tissues.Biochem J. 1988; 251: 677-683Crossref PubMed Scopus (126) Google Scholar The protein products of these genes are differentially expressed during development and display distinct tissue specificity. These three isoforms can randomly associate to form homo- and hetero-oligomers, such that the mature PFK is a tetrameric enzyme complex of ∼340 kd.4Vora S Seaman C Durham S Piomelli S Isozymes of human phosphofructokinase: identification and subunit structural characterization of a new system.Proc Natl Acad Sci USA. 1980; 77: 62-66Crossref PubMed Scopus (75) Google Scholar, 5Vora S Isozymes of human phosphofructokinase in blood cells and cultured cell lines: molecular and genetic evidence for a trigenic system.Blood. 1981; 57: 724-732Crossref PubMed Google Scholar Mature skeletal muscle expresses only the M subunit, and, therefore, contains exclusively the homotetramer M4. On the other hand, liver contains only the homotetramer L4. Erythrocytes express both the M and the L subunits, and random oligomerization gives rise to five isoenzymes, the homotetramers M4 and L4, and the three hybrid forms.4Vora S Seaman C Durham S Piomelli S Isozymes of human phosphofructokinase: identification and subunit structural characterization of a new system.Proc Natl Acad Sci USA. 1980; 77: 62-66Crossref PubMed Scopus (75) Google Scholar, 6Vora S Wims LA Durham S Morrison SL Production and characterization of monoclonal antibodies to the subunits of human phosphofructokinase: new tools for the immunochemical and genetic analyses of isozymes.Blood. 1981; 58: 823-829PubMed Google ScholarAn inherited deficiency of the PFK-M subunit (glycogenosis type VII)7Tarui S Okuno G Ikura Y Tanaka T Suda M Phosphofructokinase deficiency in skeletal muscle, a new type of glycogenosis.Biochem Biophys Res Commun. 1965; 19: 517-523Crossref PubMed Scopus (268) Google Scholar causes a syndrome, characterized by hemolysis, and by a significant myopathy with skeletal muscle weakness and cramps, exercise intolerance, and myoglobinuria.8Vora S Davidson M Seaman C Miranda AF Noble NA Tanaka KR Frenkel EP DiMauro S Heterogeneity of the molecular lesions in inherited phosphofructokinase deficiency.J Clin Invest. 1983; 72: 1995-2006Crossref PubMed Scopus (55) Google Scholar The observed clinical symptoms in patients with PFK-M deficiency reflect the lack of PFK-M in muscle and the partial reduction of the enzyme in erythrocytes.An increasing body of evidences pinpoints the association of PFK-M with cytoskeletal elements and with proteins involved in signal transduction processes, suggesting that PFK-M activity could be regulated and co-ordinated with other cellular processes in a very complex manner. In skeletal muscle, PFK-M binds to, and is inhibited by, tubulin and microtubules, indicating that the cytoskeleton may play a role in controlling the speed of glycolysis.9Lehotzky A Telegdi M Liliom K Ovadi J Interaction of phosphofructokinase with tubulin and microtubules. Quantitative evaluation of the mutual effects.J Biol Chem. 1993; 268: 10888-10894Abstract Full Text PDF PubMed Google Scholar Moreover, PFK-M was shown to form a complex with creatine kinase at the sarcomeric I-band of skeletal muscle, and this coupling is thought to increase the efficiency of cellular metabolism.10Kraft T Hornemann T Stolz M Nier V Wallimann T Coupling of creatine kinase to glycolytic enzymes at the sarcomeric I-band of skeletal muscle: a biochemical study in situ.J Muscle Res Cell Motil. 2000; 21: 691-703Crossref PubMed Scopus (56) Google Scholar In the myocardium, PFK-M interacts with phospholipase A2, suggesting a coordinated regulation of phospholipolysis and glycolysis.11Hazen SL Gross RW The specific association of a phosphofructokinase isoform with myocardial calcium-independent phospholipase A2. Implications for the coordinated regulation of phospholipolysis and glycolysis.J Biol Chem. 1993; 268: 9892-9900Abstract Full Text PDF PubMed Google Scholar PFK-M serves as a substrate for receptor tyrosine kinases, such as the insulin receptor (Ins-R) and epidermal growth factor-receptor (EGF-R) tyrosine kinases.12Reiss N Kanety H Schlessinger J Five enzymes of the glycolytic pathway serve as substrates for purified epidermal-growth-factor-receptor kinase.Biochem J. 1986; 239: 691-697Crossref PubMed Scopus (53) Google Scholar, 13Sale EM White MF Kahn CR Phosphorylation of glycolytic and gluconeogenic enzymes by the insulin receptor kinase.J Cell Biochem. 1987; 33: 15-26Crossref PubMed Scopus (30) Google Scholar In addition, PFK-M was found to be associated with neuronal nitric oxide synthase in brain and skeletal muscle.14Firestein BL Bredt DS Interaction of neuronal nitric-oxide synthase and phosphofructokinase-M.J Biol Chem. 1999; 274: 10545-10550Crossref PubMed Scopus (65) Google Scholar This interaction might be functionally relevant, as nitric oxide, the product of nitric oxide synthase activity, can regulate energy metabolism in normal muscle by stimulating exercise-induced glucose transport.15Roberts CK Barnard RJ Jasman A Balon TW Acute exercise increases nitric oxide synthase activity in skeletal muscle.Am J Physiol. 1999; 277: E390-E394PubMed Google ScholarThroughout the last decade, an emerging role in orchestrating different signaling pathways has been attributed to 50- to 100-nm membrane invaginations of the plasma membrane, termed plasmalemmal caveolae.16Lisanti MP Scherer P Tang ZL Sargiacomo M Caveolae, caveolin and caveolin-rich membrane domains: a signalling hypothesis.Trends Cell Biol. 1994; 4: 231-235Abstract Full Text PDF PubMed Scopus (587) Google Scholar, 17Smart EJ Graf GA McNiven MA Sessa WC Engelman JA Scherer PE Okamoto T Lisanti MP Caveolins, liquid-ordered domains, and signal transduction.Mol Cell Biol. 1999; 19: 7289-7304Crossref PubMed Scopus (918) Google Scholar, 18Galbiati F Razani B Lisanti MP Emerging themes in lipid rafts and caveolae.Cell. 2001; 106: 403-411Abstract Full Text Full Text PDF PubMed Scopus (514) Google Scholar Several proteins involved in signal transduction, including nitric oxide synthase isoforms, epidermal growth factor receptor, and insulin receptor, have been found to be concentrated in caveolar membrane domains. Compartmentalization within these cellular organelles appears to be essential in the regulation of the activation state of certain signaling molecules. The main structural elements of caveolae are a family of integral membrane proteins, termed caveolins.19Glenney JR Soppet D Sequence and expression of caveolin, a protein component of caveolae plasma membrane domains phosphorylated on tyrosine in RSV-transformed fibroblasts.Proc Natl Acad Sci USA. 1992; 89: 10517-10521Crossref PubMed Scopus (339) Google Scholar In mammals, the caveolin gene family is composed of three members, termed caveolin (Cav)-1, Cav-2, and Cav-3.19Glenney JR Soppet D Sequence and expression of caveolin, a protein component of caveolae plasma membrane domains phosphorylated on tyrosine in RSV-transformed fibroblasts.Proc Natl Acad Sci USA. 1992; 89: 10517-10521Crossref PubMed Scopus (339) Google Scholar, 20Scherer PE Okamoto T Chun M Nishimoto I Lodish HF Lisanti MP Identification, sequence and expression of caveolin-2 defines a caveolin gene family.Proc Natl Acad Sci USA. 1996; 93: 131-135Crossref PubMed Scopus (490) Google Scholar, 21Tang ZL Scherer PE Okamoto T Song K Chu C Kohtz DS Nishimoto I Lodish HF Lisanti MP Molecular cloning of caveolin-3, a novel member of the caveolin gene family expressed predominantly in muscle.J Biol Chem. 1996; 271: 2255-2261Crossref PubMed Scopus (606) Google Scholar, 22Way M Parton R M-caveolin, a muscle-specific caveolin-related protein.FEBS Lett. 1995; 376: 108-112Abstract Full Text PDF PubMed Scopus (260) Google Scholar Cav-1 and Cav-2 have similar tissue distributions, and are highly co-expressed in adipocytes, endothelial cells, pneumocytes, and fibroblasts,23Scherer PE Lewis RY Volonte D Engelman JA Galbiati F Couet J Kohtz DS van Donselaar E Peters P Lisanti MP Cell-type and tissue-specific expression of caveolin-2. Caveolins 1 and 2 co-localize and form a stable hetero-oligomeric complex in vivo.J Biol Chem. 1997; 272: 29337-29346Crossref PubMed Scopus (464) Google Scholar whereas Cav-3 is muscle-specific, and is highly expressed in skeletal, cardiac, and smooth muscle cells.21Tang ZL Scherer PE Okamoto T Song K Chu C Kohtz DS Nishimoto I Lodish HF Lisanti MP Molecular cloning of caveolin-3, a novel member of the caveolin gene family expressed predominantly in muscle.J Biol Chem. 1996; 271: 2255-2261Crossref PubMed Scopus (606) Google Scholar, 24Song KS Scherer PE Tang Z-L Okamoto T Li S Chafel M Chu C Kohtz DS Lisanti MP Expression of caveolin-3 in skeletal, cardiac, and smooth muscle cells. Caveolin-3 is a component of the sarcolemma and co-fractionates with dystrophin and dystrophin-associated glycoproteins.J Biol Chem. 1996; 271: 15160-15165Crossref PubMed Scopus (607) Google Scholar Several studies have shown that Cav-1 and Cav-3, but not Cav-2, can induce the formation of the caveolae organelles, by a mechanism that involves their self-oligomerization properties.25Sargiacomo M Scherer PE Tang Z-L Kubler E Song KS Sanders MC Lisanti MP Oligomeric structure of caveolin: implications for caveolae membrane organization.Proc Natl Acad Sci USA. 1995; 92: 9407-9411Crossref PubMed Scopus (475) Google Scholar, 26Monier S Parton RG Vogel F Behlke J Henske A Kurzchalia T VIP21-caveolin, a membrane protein constituent of the caveolar coat, oligomerizes in vivo and in vitro.Mol Biol Cell. 1995; 6: 911-927Crossref PubMed Scopus (397) Google Scholar, 27Galbiati F Engelman JA Volonte D Zhang XL Minetti C Li M Hou HJ Kneitz B Edelmann W Lisanti MP Caveolin-3 null mice show a loss of caveolae, changes in the microdomain distribution of the dystrophin-glycoprotein complex, and T-tubule abnormalities.J Biol Chem. 2001; 276: 21425-21433Crossref PubMed Scopus (360) Google Scholar, 28Razani B Engelman JA Wang XB Schubert W Zhang XL Marks CB Macaluso F Russell RG Li M Pestell RG Di Vizio D Hou HJ Kneitz B Lagaud G Christ GJ Edelmann W Lisanti MP Caveolin-1 null mice are viable but show evidence of hyperproliferative and vascular abnormalities.J Biol Chem. 2001; 276: 38121-38138Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar, 29Razani B Wang XB Engelman JA Battista M Lagaud G Zhang XL Kneitz B Hou Jr, H Christ GJ Edelmann W Lisanti MP Caveolin-2-deficient mice show evidence of severe pulmonary dysfunction without disruption of caveolae.Mol Cell Biol. 2002; 22: 2329-2344Crossref PubMed Scopus (252) Google ScholarCav-3 and muscle caveolae appear to have specialized roles in skeletal muscle cells, other than cellular signaling compartmentalization. Immunohistochemical studies have shown that Cav-3 is localized at the plasma membrane of skeletal muscle fibers (sarcolemma).24Song KS Scherer PE Tang Z-L Okamoto T Li S Chafel M Chu C Kohtz DS Lisanti MP Expression of caveolin-3 in skeletal, cardiac, and smooth muscle cells. Caveolin-3 is a component of the sarcolemma and co-fractionates with dystrophin and dystrophin-associated glycoproteins.J Biol Chem. 1996; 271: 15160-15165Crossref PubMed Scopus (607) Google Scholar Cav-3 expression is greatly induced during the differentiation of myoblasts to myotubes, such that fully differentiated skeletal muscle fibers show a high content of Cav-3 and Cav-3-generated caveolae.22Way M Parton R M-caveolin, a muscle-specific caveolin-related protein.FEBS Lett. 1995; 376: 108-112Abstract Full Text PDF PubMed Scopus (260) Google Scholar, 24Song KS Scherer PE Tang Z-L Okamoto T Li S Chafel M Chu C Kohtz DS Lisanti MP Expression of caveolin-3 in skeletal, cardiac, and smooth muscle cells. Caveolin-3 is a component of the sarcolemma and co-fractionates with dystrophin and dystrophin-associated glycoproteins.J Biol Chem. 1996; 271: 15160-15165Crossref PubMed Scopus (607) Google Scholar At the level of the plasma membrane, Cav-3 associates with members of a protein complex that is thought to confer structural stability to the muscle cell membrane, the dystrophin-glycoprotein complex.24Song KS Scherer PE Tang Z-L Okamoto T Li S Chafel M Chu C Kohtz DS Lisanti MP Expression of caveolin-3 in skeletal, cardiac, and smooth muscle cells. Caveolin-3 is a component of the sarcolemma and co-fractionates with dystrophin and dystrophin-associated glycoproteins.J Biol Chem. 1996; 271: 15160-15165Crossref PubMed Scopus (607) Google Scholar, 30Sotgia F Lee JK Das K Bedford M Petrucci TC Macioce P Sargiacomo M Bricarelli FD Minetti C Sudol M Lisanti MP Caveolin-3 directly interacts with the C-terminal tail of beta-dystroglycan. Identification of a central WW-like domain within caveolin family members.J Biol Chem. 2000; 275: 38048-38058Crossref PubMed Scopus (186) Google Scholar Cav-3 and dystrophin competitively bind to the same site on β-dystroglycan, suggesting that Cav-3 may have a role in regulating the membrane recruitment of dystrophin, and in the dynamic assembly of the dystrophin-glycoprotein complex.30Sotgia F Lee JK Das K Bedford M Petrucci TC Macioce P Sargiacomo M Bricarelli FD Minetti C Sudol M Lisanti MP Caveolin-3 directly interacts with the C-terminal tail of beta-dystroglycan. Identification of a central WW-like domain within caveolin family members.J Biol Chem. 2000; 275: 38048-38058Crossref PubMed Scopus (186) Google Scholar Certain signaling molecules (such as Gi2α, Gβγ, c-Src, and other Src family kinases) are found to be enriched in muscle-derived caveolar membranes, corroborating the role of muscle cell caveolae in the compartmentalization and modulation of signal transduction processes.24Song KS Scherer PE Tang Z-L Okamoto T Li S Chafel M Chu C Kohtz DS Lisanti MP Expression of caveolin-3 in skeletal, cardiac, and smooth muscle cells. Caveolin-3 is a component of the sarcolemma and co-fractionates with dystrophin and dystrophin-associated glycoproteins.J Biol Chem. 1996; 271: 15160-15165Crossref PubMed Scopus (607) Google Scholar In addition, Cav-3 interacts with nitric oxide synthase in skeletal muscle fibers,31Garcia-Cardena G Martasek P Siler-Masters BS Skidd PM Couet JC Li S Lisanti MP Sessa WC Dissecting the interaction between nitric oxide synthase (NOS) and caveolin: functional significance of the NOS caveolin binding domain in vivo.J Biol Chem. 1997; 272: 25437-25440Crossref PubMed Scopus (690) Google Scholar, 32Venema VJ Ju H Zou R Venema RC Interaction of neuronal nitric-oxide synthase with caveolin-3 in skeletal muscle. Identification of a novel caveolin scaffolding/inhibitory domain.J Biol Chem. 1997; 272: 28187-28190Crossref PubMed Scopus (216) Google Scholar a molecule that is important for the regulation of muscle contractility and exercise-induced glucose uptake.An important role for Cav-3 in muscle physiology was clearly demonstrated by the finding that mutations in the Cav-3 gene are responsible for an autosomal dominant form of limb girdle muscular dystrophy-1C.33Minetti C Sotgia F Bruno C Scartezzini P Broda P Bado M Masetti E Mazzocco M Egeo A Donati MA Volonte D Galbiati F Cordone G Bricarelli FD Lisanti MP Zara F Mutations in the caveolin-3 gene cause autosomal dominant limb-girdle muscular dystrophy.Nat Genet. 1998; 18: 365-368Crossref PubMed Scopus (487) Google Scholar The main clinical features of these patients include calf hypertrophy and mild-to-moderate muscle weakness. After this initial report, other mutations in the Cav-3 gene have been associated with different clinical phenotypes, including idiopathic hyperCKemia, rippling muscle disease, and distal myopathy.34Carbone I Bruno C Sotgia F Bado M Broda P Masetti E Panella A Zara F Bricarelli FD Cordone G Lisanti MP Minetti C Mutation in the CAV3 gene causes partial caveolin-3 deficiency and hyperCKemia.Neurology. 2000; 54: 1373-1376Crossref PubMed Scopus (123) Google Scholar, 35Betz RC Schoser BG Kasper D Ricker K Ramirez A Stein V Torbergsen T Lee YA Nothen MM Wienker TF Malin JP Propping P Reis A Mortier W Jentsch TJ Vorgerd M Kubisch C Mutations in CAV3 cause mechanical hyperirritability of skeletal muscle in rippling muscle disease.Nat Genet. 2001; 28: 218-219Crossref PubMed Scopus (164) Google Scholar, 36Tateyama M Aoki M Nishino I Hayashi YK Sekiguchi S Shiga Y Takahashi T Onodera Y Haginoya K Kobayashi K Iinuma K Nonaka I Arahata K Itoyama Y Itoyoma Y Mutation in the caveolin-3 gene causes a peculiar form of distal myopathy.Neurology. 2002; 58: 323-325Crossref PubMed Scopus (86) Google Scholar These phenotypes all share the down-regulation, to varying degrees (between 70 to 95%), of Cav-3 protein expression levels.The role of Cav-3 in skeletal muscle functioning was further enlightened by the observation that PFK-M forms a tight complex with Cav-3 under certain metabolic conditions.37Scherer PE Lisanti MP Association of phosphofructokinase-M with caveolin-3 in differentiated skeletal myotubes: dynamic regulation by extracellular glucose and intracellular metabolites.J Biol Chem. 1997; 272: 20698-20705Crossref PubMed Scopus (51) Google Scholar In C2C12 cells, as well as in skeletal muscle tissue lysates, the Cav-3/PFK-M interaction is favored by high concentrations of extracellular glucose, and is stimulated by known allosteric activators of PFK activity.37Scherer PE Lisanti MP Association of phosphofructokinase-M with caveolin-3 in differentiated skeletal myotubes: dynamic regulation by extracellular glucose and intracellular metabolites.J Biol Chem. 1997; 272: 20698-20705Crossref PubMed Scopus (51) Google Scholar As Cav-3 associates with the enzymatically active form of PFK-M, these findings indicate that interaction of PFK-M with Cav-3 would be expected to recruit PFK-M to the muscle plasma membrane, and concentrate it within caveolar membrane domains. However, direct evidence for Cav-3-mediated recruitment of PFK-M to the plasma membrane is lacking. Thus, this hypothesis remains to be tested experimentally.Here, we directly demonstrate that recombinant expression of Cav-3 regulates the subcellular distribution of PFK-M. We show that Cav-3 expression recruits PFK-M to the plasma membrane and caveolin-containing lipid raft microdomains. In contrast, expression of Cav-3 mutants that cause limb girdle muscular dystrophy and other muscle diseases prevents the plasma membrane targeting of PFK-M and induces the degradation of PFK-M by a proteasomal pathway. Finally, analysis of skeletal muscle tissue from Cav-3-deficient mice clearly demonstrates the importance of Cav-3 in the regulation of PFK-M behavior. Skeletal muscles from Cav-3-null mice show unperturbed expression levels of PFK-M, but significant changes in the subcellular localization of PFK-M. More specifically, PFK-M is no longer recruited to the plasma membrane, and is specifically excluded from lipid rafts/caveolar microdomains in Cav-3-null mice. Taken together, these results highlight the central role of muscle caveolae in the control of energy metabolism in skeletal muscle fibers and provide insight into the molecular mechanisms underlying muscle diseases in which Cav-3 expression is down-regulated.Materials and MethodsMaterialsAntibodies and their sources were as follows: anti-V5 monoclonal antibody (mAb) (Invitrogen, Carlsbad, CA); anti-Cav-3 mAb (clone 26)24Song KS Scherer PE Tang Z-L Okamoto T Li S Chafel M Chu C Kohtz DS Lisanti MP Expression of caveolin-3 in skeletal, cardiac, and smooth muscle cells. Caveolin-3 is a component of the sarcolemma and co-fractionates with dystrophin and dystrophin-associated glycoproteins.J Biol Chem. 1996; 271: 15160-15165Crossref PubMed Scopus (607) Google Scholar (gift of Dr. Roberto Campos-Gonzalez; BD Transduction Laboratories, Inc.); rabbit anti-PFK-M polyclonal antibody (pAb) and guinea pig anti-PFK-M pAb (generated by Dr. Robert G. Kemp, University of Health Sciences, The Chicago Medical School, North Chicago, IL); anti-Cav-3 pAb (Affinity Bioreagents, Inc.); anti-Cav-1 pAb (Santa Cruz Biotechnology, Santa Cruz, CA); anti-actin mAb (clone AC-40) (Sigma Chemical Co., St. Louis, MO); anti-β-actin mAb (clone AC-15) (Sigma); proaerolysin (Protox Biotech, Inc., Victoria, Canada); anti-aerolysin polyclonal Ab (gift of Dr. J. Thomas Buckley, University of Victoria, Canada). A variety of other reagents were purchased commercially, as follows: cell-culture reagents were from Life Technologies, Inc. Grand Island, NY; the Effectene transfection reagent was from Qiagen, Valencia, CA; glutathione-agarose beads were from Amersham, Arlington Heights, IL; the proteasomal inhibitor, MG-132, was from Calbiochem, La Jolla, CA.Expression VectorsThe cDNAs encoding Cav-3 and Cav-3 mutants (Cav-3 P104L, Cav-3 ΔTFT, and Cav-3 R26Q) were subcloned into pCAGGS, a mammalian expression vector driven by the cytomegalovirus promoter.38Galbiati F Volonte D Minetti C Chu JB Lisanti MP Phenotypic behavior of caveolin-3 mutations that cause autosomal dominant limb girdle muscular dystrophy (LGMD-1C).J Biol Chem. 1999; 274: 25632-25641Crossref PubMed Scopus (136) Google Scholar, 39Sotgia F Woodman SE Bonuccelli G Capozza F Minetti C Scherer PE Lisanti MP Phenotypic behavior of caveolin-3 R26Q, a mutant associated with hyperCKemia, distal myopathy, and rippling muscle disease.Am J Physiol Cell Physiol. 2003; 285: C1150-C1160Crossref PubMed Scopus (35) Google Scholar The cDNAs encoding human PFK-M, PFK-B, and PFK-P in the pEF6/V5 TOPO vector under the control of the human EF-1α promoter were purchased from Invitrogen (Genestorm Clones).Cell Culture and TransfectionCos-7 and 293T cells were grown in DME supplemented with 10% fetal bovine serum, 2 mmol/L glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin (Life Technologies, Inc.). Cells (∼40 to 50% confluent) were transiently transfected using the Effectene transfection reagent, as per the manufacturer's instructions, and analyzed 36 to 48 hours after transfection.Construction and Purification of GST Fusion ProteinsGST-Cav-3 and GST-Cav-1 expression constructs were as previously described.30Sotgia F Lee JK Das K Bedford M Petrucci TC Macioce P Sargiacomo M Bricarelli FD Minetti C Sudol M Lisanti MP Caveolin-3 directly interacts with the C-terminal tail of beta-dystroglycan. Identification of a central WW-like domain within caveolin family members.J Biol Chem. 2000; 275: 38048-38058Crossref PubMed Scopus (186) Google Scholar Briefly, Cav-3 (residues 34 to 129) and Cav-1 (residues 1 to 178) were amplified and subcloned into the pGEX-4T vector. GST fusion protein constructs were transformed into Escherichia coli (BL21 strain; Novagen, Inc., Madison, WI). After induction of expression through addition of 0.5 mmol/L of isopropyl-β-d-thio-galactoside (Sigma), GST fusion proteins were affinity-purified on glutathione-agarose beads, using the detergent Sarcosyl for initial solubilization.GST Pull-Down AssaysThe pull-down assay using GST alone or GST-Cav-1/3 fusion proteins was essentially as previously described.30Sotgia F Lee JK Das K Bedford M Petrucci TC Macioce P Sargiacomo M Bricarelli FD Minetti C Sudol M Lisanti MP Caveolin-3 directly interacts with the C-terminal tail of beta-dystroglycan. Identification of a central WW-like domain within caveolin family members.J Biol Chem. 2000; 275: 38048-38058Crossref PubMed Scopus (186) Google Scholar Briefly, 293T cells transiently overexpressing V5-tagged PFK-M were lysed in RIPA buffer [10 mmol/L Tris-HCl, pH 7.4, 300 mmol/L NaCl, 1% Triton X-100, 0.1% sodium dodecyl sulfate (SDS), 1% sodium deoxycholate]. Precleared lysates were then diluted in Tween buffer (50 mmol/L Tris-HCl, pH 7.4, 1 mmol/L ethylenediaminetetraacetic acid, 100 mmol/L NaCl, 0.1% Tween-20, 1 mmol/L dithiothreitol, and protease inhibitors) and added to ∼100 μl of an equalized bead volume for overnight incubation at 4°C. After binding, the beads were extensively washed with phosphate-buffered saline (six times). Finally, the beads were resuspended in 3× sample buffer, boiled, and subjected to SDS-polyacrylamide gel electrophoresis (PAGE).Immunoblot AnalysisTransfected cells were washed twice with phosphate-buffered saline (PBS), and lysed with hot sample buffer containing dithiothreitol. To prepare tissue lysates, mouse skeletal muscle tissue was harvested, minced with scissors, homogenized in a Polytron tissue grinder for 30 seconds at a medium range speed, using boiling lysis buffer (10 mmol/L Tris, pH 8; 1% SDS) containing protease inhibitors (Boehringer Mannheim, Indianapolis, IN). Protein concentrations were quantified using the BCA reagent (Pierce, Rockford, IL) and the volume required for 10 μg of protein was determined. Samples were separated by SDS-PAGE (12.5 or 10% acrylamide) and transferred to nitrocellulose. The nitrocellulose membranes were stained with Ponceau S (to visualize protein bands) followed by immunoblot analysis. All subsequent wash buffers contained 10 mmol/L Tris, pH 8.0, 150 mmol/L NaCl, 0.05% Tween-20, which was supplemented with 1% bovine serum albumin (BSA) and 4% nonfat dry milk (Carnation) for the blocking solution and 1% BSA for the antibody diluent. Horseradish peroxidase-conjugated secondary antibodies were used to visu
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