Actin Directly Interacts with Phospholipase D, Inhibiting Its Activity

Mammalian phospholipase D (PLD) plays a key role in several signal transduction pathways and is involved in many diverse functions. To elucidate the complex molecular regulation of PLD, we investigated PLD-binding proteins obtained from rat brain extract. Here we report that a 43-kDa protein in the rat brain, β-actin, acts as a major PLD2 direct-binding protein as revealed by peptide mass fingerprinting in combination with matrix-assisted laser desorption ionization/time-of-flight mass spectrometry. We also determined that the region between amino acids 613 and 723 of PLD2 is required for the direct binding of β-actin, using bacterially expressed glutathione S-transferase fusion proteins of PLD2 fragments. Intriguingly, purified β-actin potently inhibited both phosphatidylinositol-4,5-bisphosphate- and oleate-dependent PLD2 activities in a concentration-dependent manner (IC50 = 5 nm). In a previous paper, we reported that α-actinin inhibited PLD2 activity in an interaction-dependent and an ADP-ribosylation factor 1 (ARF1)-reversible manner (Park, J. B., Kim, J. H., Kim, Y., Ha, S. H., Kim, J. H., Yoo, J.-S., Du, G., Frohman, M. A., Suh, P.-G., and Ryu, S. H. (2000) J. Biol. Chem. 275, 21295–21301). In vitro binding analyses showed that β-actin could displace α-actinin binding to PLD2, demonstrating independent interaction between cytoskeletal proteins and PLD2. Furthermore, ARF1 could steer the PLD2 activity in a positive direction regardless of the inhibitory effect of β-actin on PLD2. We also observed that β-actin regulates PLD1 and PLD2 with similar binding and inhibitory potencies. Immunocytochemical and co-immunoprecipitation studies demonstrated the in vivointeraction between the two PLD isozymes and actin in cells. Taken together, these results suggest that the regulation of PLD by cytoskeletal proteins, β-actin and α-actinin, and ARF1 may play an important role in cytoskeleton-related PLD functions. Mammalian phospholipase D (PLD) plays a key role in several signal transduction pathways and is involved in many diverse functions. To elucidate the complex molecular regulation of PLD, we investigated PLD-binding proteins obtained from rat brain extract. Here we report that a 43-kDa protein in the rat brain, β-actin, acts as a major PLD2 direct-binding protein as revealed by peptide mass fingerprinting in combination with matrix-assisted laser desorption ionization/time-of-flight mass spectrometry. We also determined that the region between amino acids 613 and 723 of PLD2 is required for the direct binding of β-actin, using bacterially expressed glutathione S-transferase fusion proteins of PLD2 fragments. Intriguingly, purified β-actin potently inhibited both phosphatidylinositol-4,5-bisphosphate- and oleate-dependent PLD2 activities in a concentration-dependent manner (IC50 = 5 nm). In a previous paper, we reported that α-actinin inhibited PLD2 activity in an interaction-dependent and an ADP-ribosylation factor 1 (ARF1)-reversible manner (Park, J. B., Kim, J. H., Kim, Y., Ha, S. H., Kim, J. H., Yoo, J.-S., Du, G., Frohman, M. A., Suh, P.-G., and Ryu, S. H. (2000) J. Biol. Chem. 275, 21295–21301). In vitro binding analyses showed that β-actin could displace α-actinin binding to PLD2, demonstrating independent interaction between cytoskeletal proteins and PLD2. Furthermore, ARF1 could steer the PLD2 activity in a positive direction regardless of the inhibitory effect of β-actin on PLD2. We also observed that β-actin regulates PLD1 and PLD2 with similar binding and inhibitory potencies. Immunocytochemical and co-immunoprecipitation studies demonstrated the in vivointeraction between the two PLD isozymes and actin in cells. Taken together, these results suggest that the regulation of PLD by cytoskeletal proteins, β-actin and α-actinin, and ARF1 may play an important role in cytoskeleton-related PLD functions. phospholipase D phosphatidylcholine phosphatidylinositol-4,5-bisphosphate ADP-ribosylation factor guanosine 5′-3′-O-(thio) triphosphate Triton X-100 polyacrylamide gel electrophoresis glutathione S-transferase 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid pheochromocytoma green fluorescent protein Mammalian phospholipase D (PLD)1 hydrolyzes phosphatidylcholine (PC) to generate phosphatidic acid and choline in response to a variety of signals, which can include hormones, neurotransmitters, and growth factors (1Exton J.H. Biochim. Biophys. Acta. 1997; 1439: 121-133Crossref Scopus (327) Google Scholar). phosphatidic acid itself has been shown to be an intracellular lipid second messenger and to be involved in multiple physiological events such as the promotion of mitogenesis, stimulation of respiratory bursts, secretory processes, actin cytoskeletal reorganization, and the activation of Raf-1 kinase and phosphatidylinositol 4-phosphate (PtdIns4P) 5-kinase isoforms in a large number of cells. These relationships suggest that agonist-induced PLD activation may play roles in multiple signaling events (2Jones D. Morgan C. Cockcroft S. Biochim. Biophys. Acta. 1999; 1439: 229-244Crossref PubMed Scopus (168) Google Scholar, 3Danniel L.W. Sciorra V.A. Ghosh S. Biochim. Biophys. Acta. 1999; 1439: 265-276Crossref PubMed Scopus (36) Google Scholar, 4Olson S.C. Lambeth J.D. Chem. Phys. Lipids. 1996; 80: 3-19Crossref PubMed Scopus (54) Google Scholar, 5Cross M.J. Roberts S. Ridley A.J. Hodgkin M.N. Stewart A. Claesson-Welsh L. 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Lee S.D. Lee T.G. Kim Y. Park J.B. Lambeth J.D. Suh P.G. Ryu S.H. 1998; PubMed Scopus Google Scholar). PLD2 also on but has a basal activity PLD1 Lambeth J.D. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google and has been that PLD2 may be with different many studies to on the relationships and the of the PLD the molecular of the regulation of the has been In the of PLD-binding may the of the complex regulatory of PLD in different cells. has been observed in many studies that PLD is in the of cells. PLD activity has been in the of that a of cytoskeletal proteins M.N. S. K. Wakelam M.J.O. Biochem. J. 1999; PubMed Scopus Google Scholar, J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus (54) Google Scholar). cytoskeletal proteins such as and have been to as in vitro S. A. Biochem. Biophys. 1998; PubMed Scopus Google Scholar, S. H. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, S. 1996; PubMed Scopus Google and agonist-induced PLD stimulation can in cytoskeletal M.J. Roberts S. Ridley A.J. Hodgkin M.N. Stewart A. Claesson-Welsh L. Wakelam M.J.O. Curr. Biol. 1996; 6: 588-597Abstract Full Text Full Text PDF PubMed Google Scholar, K.S. Exton J.H. J. Biol. 1993; PubMed Scopus Google Scholar, K.S. Exton J.H. Biochem. J. PubMed Scopus Google Scholar, Sung T.C. J. Y. D. Morris A.J. Frohman M.A. Curr. Biol. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). Furthermore, we reported that α-actinin, an also to PLD2 to activity J.B. Kim J.H. Kim S.H. Kim J.H. J. G. Frohman M.A. Suh P.G. Ryu S.H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). is a of a regulatory between PLD and the actin In we for the that β-actin, a major cytoskeletal regulates PLD by direct We also the relationships and of of ARF1 and cytoskeletal proteins on PLD using PLD2 as a and the results obtained suggest for the regulation of PLD by these The and from and from from obtained from and and from from protein and from obtained from obtained from that both PLD1 and PLD2 as J.B. Kim J.H. Kim S.H. Kim J.H. J. G. Frohman M.A. Suh P.G. Ryu S.H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). by A. of PLD2 and by A. Frohman of PLD1 and PLD2 purified from of by as J.H. Kim Y. Lee S.D. Lambeth J.D. Suh P.G. Ryu S.H. 1999; PubMed Scopus Google Scholar). in 1 1 1 and using a for 1 the to in the brain determined using the by Biochem. PubMed Scopus Google Scholar). on protein with purified PLD2 for a the with and The brain of with the for the proteins with a and by The as J.B. Kim J.H. Kim S.H. Kim J.H. J. G. Frohman M.A. Suh P.G. Ryu S.H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In the 43-kDa protein co-immunoprecipitation from rat brain by and the to and with for The of the obtained determined with a mass in the in peptide with mass on the amino acid and the mass of the of the for a protein β-actin purified from rat brain as D. C. J. Mol. Biol. PubMed Scopus Google and to be by ARF1 expressed in and purified J.D. D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The of PLD2 These PLD2 the of the β-actin by Kim the of the and the to the the fusion proteins J. T. Scholar). with the fusion proteins, and the the fusion proteins expressed purified by C. Kim Chung J.K. Frohman M.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google using in a on The two PLD isozymes and the of PLD2 to PLD These with the of purified β-actin ARF1 for of the PLD-binding in the PLD and the in the a polyacrylamide The in of the with β-actin also in the using α-actinin binding similar to for β-actin In PLD2 with α-actinin and β-actin for in the PLD and the with the as J.B. Kim J.H. Kim S.H. Kim J.H. J. G. Frohman M.A. Suh P.G. Ryu S.H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). PLD activity by choline from phosphatidylcholine H.A. Gutowski S. Moomaw C.R. Slaughter C. Sternweis P.C. Cell. 1993; 75: 1137-1144Abstract Full Text PDF PubMed Scopus (821) Google with In the for in a the PLD the PLD and of of and of in a of PLD activity as J.H. Kim Y. Lee S.D. Lambeth J.D. Suh P.G. Ryu S.H. 1999; PubMed Scopus Google Scholar). In 5 of and of to a and The of in the The for 1 and the by the of of 1 and 1 of a the of in of the by by for 5 in a PubMed Scopus Google by and to by using the in and the with which by with as with using an to The to the of PLD2 in S.D. Lee Kim J.H. Kim Y. Suh P.G. Ryu S.H. J. 75: PubMed Scopus Google Scholar). in with and To PLD2, the in the in a growth of with in a and the of PLD a of 1 in and using as J.H. Lee Kim Y. Lee S.D. Suh P.G. Ryu S.H. J. 1999; Google Scholar). in the presence of PLD1 with PLD 1 1 and 5 the for with and for 1 The of with on protein for the with a polyacrylamide gel for with the and the of PLD2 by in PLD The for 1 and the in the and to as as Y. Lee Kim J.H. Lee Suh P.G. Ryu S.H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In on in the presence of with and with for with and with and for the with to PLD The with and in with and for 1 to PLD2 and actin To in Y. Kim Lee S.D. Lee T.G. Kim J.H. Park J.B. S.K. Suh P.G. Ryu S.H. Biochim. 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C. J. Mol. Biol. PubMed Scopus Google and with the shown in the demonstrated that β-actin with PLD2. To the PLD2 sequence involved in the β-actin we the fusion proteins shown in and for to to purified acids to be the region that potently to β-actin that the region of the protein between amino acids 613 and 723 may be important for the direct interaction with We PLD activity to the effect of β-actin on PLD2. shown in the activity of PLD2 inhibited in a β-actin concentration-dependent β-actin purified from rat brain, the required for 5 To the inhibitory effect of β-actin on PLD2 we and purified a for of PLD we observed that an inhibitory effect that similar to that of the β-actin purified from rat To the that the PLD2 activity be by in the we also a PLD2 activity in the of We reported that PLD2 could be activated by in the of J.H. Kim Y. Lee S.D. Lambeth J.D. Suh P.G. Ryu S.H. 1999; PubMed Scopus Google Scholar). shown in PLD2 activated by inhibited by the of β-actin purified from rat brain with similar inhibitory The inhibitory of β-actin, the of the to that observed the that Taken together, these results suggest that the inhibitory effect of β-actin on PLD2 be by direct In a previous we that α-actinin to the region acids of PLD2 and activity of the J.B. Kim J.H. Kim S.H. Kim J.H. J. G. Frohman M.A. Suh P.G. Ryu S.H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). To the between the two cytoskeletal proteins, β-actin and α-actinin, in of PLD2 in vitro binding that an in β-actin α-actinin binding to PLD2 in a and concentration-dependent In the binding of β-actin to PLD2 the of α-actinin to PLD2. To the that of the binding on the two proteins, we an α-actinin binding region of PLD2 In vitro binding showed that β-actin to with an with that of PLD2 that β-actin to the α-actinin binding region of PLD2. To of we also a PLD activity and the inhibitory of β-actin on and shown in 5 β-actin potently inhibited the activities of both and to similar These results that β-actin may PLD2 activity by with a the α-actinin binding region and suggest that β-actin and α-actinin on PLD2 in a to the of PLD2 activity by β-actin could be by we reported that the α-actinin inhibited PLD2 activity could be by ARF1 and that by the of α-actinin from PLD2 by ARF1 binding J.B. Kim J.H. Kim S.H. Kim J.H. J. G. Frohman M.A. Suh P.G. Ryu S.H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). we the effect of ARF1 in by the of ARF1 in the PLD activity in the presence and of purified shown in β-actin inhibited PLD2 could be activated by the of ARF1, and independent of the effect of β-actin to PLD2. that activation may be by but by different of binding between PLD2 and ARF1 To elucidate the of of ARF1 on the we ARF1 in the presence of PLD2 and that the of ARF1 to in the presence of ARF1 Taken together, these results suggest that β-actin to PLD2 of ARF1 and that PLD2 be by a of of ARF1 that from that of The that actin may with PLD2 the by using a co-immunoprecipitation on which to the of PLD2 S.D. Lee Kim J.H. Kim Y. Suh P.G. Ryu S.H. J. 75: PubMed Scopus Google Scholar). shown in a very low of PLD2 expressed in in the presence of which we of the of PLD2. with PLD2 from of the in the of demonstrating a interaction between actin and PLD2 in In these the of PLD2 the of actin To PLD2 with the actin in the with the shown in the PLD2 to be the of the and of actin with showed that the PLD2 with in the cells. Taken together, these results suggest that actin regulation of PLD2 direct interaction the actin Mammalian PLD has two PLD1 and PLD2, that have a sequence homology of To the effect of purified β-actin on these two PLD vitro binding that β-actin with both PLD isozymes with the To the inhibitory effect of β-actin on PLD we purified β-actin and PLD isozymes in a PLD activity in the presence of In we the activity of both PLD isozymes activated by ARF1 to the effect of β-actin the that β-actin inhibited PLD1 and PLD2 activity in a concentration-dependent To actin also a complex with PLD1 in we a PLD1 in cells. The to co-immunoprecipitation and shown in actin in of the but in of the that actin with PLD1 in cells. We also in these that actin with PLD2 as shown in To the interaction between PLD isozymes and the actin in we cells. shown in with the two PLD which an between PLD and the actin Taken together, these results suggest that β-actin may the activities of both PLD isozymes direct interaction in these cells. that that PLD activity is to the cytoskeletal and may be involved in actin cytoskeletal in the regulatory of PLD in the actin has we to a of PLD-binding proteins that could be to the regulation of In paper, we PLD2 as a and investigated proteins using a co-immunoprecipitation with rat brain a β-actin, a major of the identified as a protein and that direct interaction between β-actin and PLD2 the activity of the In β-actin, α-actinin, ARF1 with PLD2 we a of for the regulation of the PLD complex by cytoskeletal proteins and PLD2 is in has been that may with in In the paper, we report for the that β-actin with PLD2 and activity and of the that actin may have a role as an by with PLD2. purified β-actin PLD2 a very low the PLD2 is by factors such as in the purified actin also PLD2 activity in a manner similar to purified β-actin the inhibitory activity of β-actin on PLD2 is by the presence of These results suggest that the by β-actin is by We also that the PLD2 is by the PLD as cytoskeleton-related proteins such as α-actinin, and S. A. Biochem. Biophys. 1998; PubMed Scopus Google Scholar, S. H. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, S. 1996; PubMed Scopus Google Scholar, J.B. Kim J.H. Kim S.H. Kim J.H. J. G. Frohman M.A. Suh P.G. Ryu S.H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Y. H. Y. S. Y. Y. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus (65) Google and proteins such as and protein C. Kim Chung J.K. Frohman M.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A. Morris 1998; PubMed Scopus Google Scholar, J.K. Kang H.S. Lee C. J.S. Kim Bae Morris A.J. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, C. Kang H.S. Chung J.K. Kim J.S. Kim Bae Morris A.J. J. Biol. 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The actin is a of actin and a large variety of proteins L. M. J. Biol. Chem. 1993; Google Scholar, G. J. Biol. Chem. Scholar). In PLD activity has been reported in the of and M.N. S. K. Wakelam M.J.O. Biochem. J. 1999; PubMed Scopus Google Scholar, J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus (54) Google demonstrating the of in interaction between PLD and the has been that several proteins such as and α-actinin as in vitro S. A. Biochem. Biophys. 1998; PubMed Scopus Google Scholar, S. H. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, J.B. Kim J.H. Kim S.H. Kim J.H. J. G. Frohman M.A. Suh P.G. Ryu S.H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). been report that has an in vivointeraction between PLD2 and the actin is the to that PLD2 is the of has been reported in Sung T.C. J. Y. D. Morris A.J. Frohman M.A. Curr. Biol. 1997; Full Text Full Text PDF PubMed Scopus Google and that is with actin co-immunoprecipitation results also a interaction between PLD2 and the actin in results suggest that the actin may be with and involved in the regulation of PLD2. In we to a for the complex regulatory between the cytoskeletal proteins, ARF1, and PLD2. that β-actin a binding acids on PLD2, which from that by α-actinin and that ARF1 share an an binding with β-actin on PLD2. reported α-actinin with the region acids of PLD2, and that ARF1 can the of α-actinin from PLD2 by a in the J.B. Kim J.H. Kim S.H. Kim J.H. J. G. Frohman M.A. Suh P.G. Ryu S.H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the regulatory of the PLD2 by ARF1 from regulation of we suggest that the regulation of PLD2 by β-actin α-actinin different binding on PLD2, that they may and that they may be activated by different of of that the of β-actin to PLD2 is similar in both and α-actinin binding region the that β-actin and α-actinin different binding and by We also suggest that the independent interaction between the two cytoskeletal proteins and PLD2 is by an interaction between β-actin and α-actinin, the interaction between actin and α-actinin have been reported to be very = C. M. C. Y. Biochem. Biophys. PubMed Scopus Google and we that to α-actinin the to the that PLD2 may in regulation by β-actin α-actinin may to the and in the and that be in a different to We suggest that the complex involved in the regulation of PLD2 by cytoskeletal proteins may be to the actin cytoskeletal required by diverse plays a major role in the regulation of PLD J.H. 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Cell. 1999; 99: 512-532Abstract Full Text Full Text PDF Scopus (699) Google reported that with PLD2 in with growth these suggest that proteins be involved in the complex of the regulation of PLD2 in the actin The mammalian PLD PLD1 and PLD2, have a sequence homology of and in in the PLD A.J. J. Frohman M.A. 1996; Full Text PDF PubMed Scopus Google Scholar, Frohman M.A. Biochem. J. 1997; PubMed Scopus Google Scholar). many previous have that the two PLD isozymes may different regulatory from the that β-actin with and both PLD isozymes with similar vitro and that actin may have a interaction and regulation for PLD in cells. is by that actin and with two PLD isozymes in cells. the of we also suggest that region the β-actin binding region of PLD2, may to the interaction and regulation of the two PLD many region is in PLD that PLD Furthermore, we suggest that the regulation of PLD by the actin may on β-actin binding to region of PLD in cells. In we have identified of the PLD-binding proteins as β-actin and that direct interaction between β-actin and PLD PLD the of we suggest that the between the actin and PLD may PLD activity in cells. stimulation by the of to the may the activity of PLD by the actin In the we to the and of regulatory in the actin