Determination of interaction sites of phospholipase D1 for RhoA (original) (raw)

Abstract

Phospholipase D (PLD) is regulated by many factors, including protein kinase C (PKC) and small G-proteins of the Rho and ADP-ribosylation factor families. Previous studies revealed that the interaction site of human PLD(1) for RhoA is located in its C-terminus, but the exact locus has not been determined. The purpose of the present study was to determine the interaction site of rat PLD(1) (rPLD(1)) with RhoA. Selection with phage display of different peptides of rPLD(1) confirmed that GTP-bound RhoA interacted with a site in the amino acid sequence 873-1024 at the C-terminus of rPLD(1). RhoA also associated with this peptide in a GTP-dependent manner in COS-7 cell lysates and the peptide inhibited RhoA stimulation of PLD activity in membranes from COS-7 cells expressing rPLD(1). A series of alanine mutations of non-conserved residues were made in this sequence, and the enzymes were expressed in COS-7 cells and checked for responses to activation of PKC, which interacts with the N-terminus of PLD(1), and also to the constitutively active V14RhoA. Mutations in the C-terminus of rPLD(1) (K946A, V950A, R955A and K962A) caused partial loss of V14RhoA stimulation, and double mutations (K946A/K962A, K946A/V950A and K962A/V950A) caused an almost total loss. Co-immunoprecipitation studies also showed that the mutated forms of rPLD(1) described above failed to bind V14RhoA compared with wild-type rPLD(1), whereas rPLD(1) with mutations outside the region K946-K962 bound V14RhoA normally. It is concluded that basic amino acids in a restricted C-terminal region of rPLD(1) are important for binding of RhoA and its activation of PLD activity.

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Selected References

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  1. Bae C. D., Min D. S., Fleming I. N., Exton J. H. Determination of interaction sites on the small G protein RhoA for phospholipase D. J Biol Chem. 1998 May 8;273(19):11596–11604. doi: 10.1074/jbc.273.19.11596. [DOI] [PubMed] [Google Scholar]
  2. Burbelo P. D., Drechsel D., Hall A. A conserved binding motif defines numerous candidate target proteins for both Cdc42 and Rac GTPases. J Biol Chem. 1995 Dec 8;270(49):29071–29074. doi: 10.1074/jbc.270.49.29071. [DOI] [PubMed] [Google Scholar]
  3. Colley W. C., Sung T. C., Roll R., Jenco J., Hammond S. M., Altshuller Y., Bar-Sagi D., Morris A. J., Frohman M. A. Phospholipase D2, a distinct phospholipase D isoform with novel regulatory properties that provokes cytoskeletal reorganization. Curr Biol. 1997 Mar 1;7(3):191–201. doi: 10.1016/s0960-9822(97)70090-3. [DOI] [PubMed] [Google Scholar]
  4. Diekmann D., Nobes C. D., Burbelo P. D., Abo A., Hall A. Rac GTPase interacts with GAPs and target proteins through multiple effector sites. EMBO J. 1995 Nov 1;14(21):5297–5305. doi: 10.1002/j.1460-2075.1995.tb00214.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Du G., Altshuller Y. M., Kim Y., Han J. M., Ryu S. H., Morris A. J., Frohman M. A. Dual requirement for rho and protein kinase C in direct activation of phospholipase D1 through G protein-coupled receptor signaling. Mol Biol Cell. 2000 Dec;11(12):4359–4368. doi: 10.1091/mbc.11.12.4359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Exton J. H. Regulation of phospholipase D. Biochim Biophys Acta. 1999 Jul 30;1439(2):121–133. doi: 10.1016/s1388-1981(99)00089-x. [DOI] [PubMed] [Google Scholar]
  7. Freeman J. L., Abo A., Lambeth J. D. Rac "insert region" is a novel effector region that is implicated in the activation of NADPH oxidase, but not PAK65. J Biol Chem. 1996 Aug 16;271(33):19794–19801. doi: 10.1074/jbc.271.33.19794. [DOI] [PubMed] [Google Scholar]
  8. Fujisawa K., Fujita A., Ishizaki T., Saito Y., Narumiya S. Identification of the Rho-binding domain of p160ROCK, a Rho-associated coiled-coil containing protein kinase. J Biol Chem. 1996 Sep 20;271(38):23022–23028. doi: 10.1074/jbc.271.38.23022. [DOI] [PubMed] [Google Scholar]
  9. Fujisawa K., Madaule P., Ishizaki T., Watanabe G., Bito H., Saito Y., Hall A., Narumiya S. Different regions of Rho determine Rho-selective binding of different classes of Rho target molecules. J Biol Chem. 1998 Jul 24;273(30):18943–18949. doi: 10.1074/jbc.273.30.18943. [DOI] [PubMed] [Google Scholar]
  10. Gottlin E. B., Rudolph A. E., Zhao Y., Matthews H. R., Dixon J. E. Catalytic mechanism of the phospholipase D superfamily proceeds via a covalent phosphohistidine intermediate. Proc Natl Acad Sci U S A. 1998 Aug 4;95(16):9202–9207. doi: 10.1073/pnas.95.16.9202. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hammond S. M., Altshuller Y. M., Sung T. C., Rudge S. A., Rose K., Engebrecht J., Morris A. J., Frohman M. A. Human ADP-ribosylation factor-activated phosphatidylcholine-specific phospholipase D defines a new and highly conserved gene family. J Biol Chem. 1995 Dec 15;270(50):29640–29643. doi: 10.1074/jbc.270.50.29640. [DOI] [PubMed] [Google Scholar]
  12. Hammond S. M., Jenco J. M., Nakashima S., Cadwallader K., Gu Q., Cook S., Nozawa Y., Prestwich G. D., Frohman M. A., Morris A. J. Characterization of two alternately spliced forms of phospholipase D1. Activation of the purified enzymes by phosphatidylinositol 4,5-bisphosphate, ADP-ribosylation factor, and Rho family monomeric GTP-binding proteins and protein kinase C-alpha. J Biol Chem. 1997 Feb 7;272(6):3860–3868. doi: 10.1074/jbc.272.6.3860. [DOI] [PubMed] [Google Scholar]
  13. Ihara K., Muraguchi S., Kato M., Shimizu T., Shirakawa M., Kuroda S., Kaibuchi K., Hakoshima T. Crystal structure of human RhoA in a dominantly active form complexed with a GTP analogue. J Biol Chem. 1998 Apr 17;273(16):9656–9666. doi: 10.1074/jbc.273.16.9656. [DOI] [PubMed] [Google Scholar]
  14. Jones D. H., Bax B., Fensome A., Cockcroft S. ADP ribosylation factor 1 mutants identify a phospholipase D effector region and reveal that phospholipase D participates in lysosomal secretion but is not sufficient for recruitment of coatomer I. Biochem J. 1999 Jul 1;341(Pt 1):185–192. [PMC free article] [PubMed] [Google Scholar]
  15. Joneson T., Bar-Sagi D. A Rac1 effector site controlling mitogenesis through superoxide production. J Biol Chem. 1998 Jul 17;273(29):17991–17994. doi: 10.1074/jbc.273.29.17991. [DOI] [PubMed] [Google Scholar]
  16. Kanfer J. N. The base exchange enzymes and phospholipase D of mammalian tissue. Can J Biochem. 1980 Dec;58(12):1370–1380. doi: 10.1139/o80-186. [DOI] [PubMed] [Google Scholar]
  17. Kodaki T., Yamashita S. Cloning, expression, and characterization of a novel phospholipase D complementary DNA from rat brain. J Biol Chem. 1997 Apr 25;272(17):11408–11413. doi: 10.1074/jbc.272.17.11408. [DOI] [PubMed] [Google Scholar]
  18. Liscovitch M., Czarny M., Fiucci G., Lavie Y., Tang X. Localization and possible functions of phospholipase D isozymes. Biochim Biophys Acta. 1999 Jul 30;1439(2):245–263. doi: 10.1016/s1388-1981(99)00098-0. [DOI] [PubMed] [Google Scholar]
  19. Liscovitch M., Czarny M., Fiucci G., Tang X. Phospholipase D: molecular and cell biology of a novel gene family. Biochem J. 2000 Feb 1;345(Pt 3):401–415. [PMC free article] [PubMed] [Google Scholar]
  20. Lopez I., Arnold R. S., Lambeth J. D. Cloning and initial characterization of a human phospholipase D2 (hPLD2). ADP-ribosylation factor regulates hPLD2. J Biol Chem. 1998 May 22;273(21):12846–12852. doi: 10.1074/jbc.273.21.12846. [DOI] [PubMed] [Google Scholar]
  21. Malcolm K. C., Elliott C. M., Exton J. H. Evidence for Rho-mediated agonist stimulation of phospholipase D in rat1 fibroblasts. Effects of Clostridium botulinum C3 exoenzyme. J Biol Chem. 1996 May 31;271(22):13135–13139. doi: 10.1074/jbc.271.22.13135. [DOI] [PubMed] [Google Scholar]
  22. Manser E., Leung T., Salihuddin H., Zhao Z. S., Lim L. A brain serine/threonine protein kinase activated by Cdc42 and Rac1. Nature. 1994 Jan 6;367(6458):40–46. doi: 10.1038/367040a0. [DOI] [PubMed] [Google Scholar]
  23. Min D. S., Exton J. H. Phospholipase D is associated in a phorbol ester-dependent manner with protein kinase C-alpha and with a 220-kDa protein which is phosphorylated on serine and threonine. Biochem Biophys Res Commun. 1998 Jul 30;248(3):533–537. doi: 10.1006/bbrc.1998.8990. [DOI] [PubMed] [Google Scholar]
  24. Min D. S., Park S. K., Exton J. H. Characterization of a rat brain phospholipase D isozyme. J Biol Chem. 1998 Mar 20;273(12):7044–7051. doi: 10.1074/jbc.273.12.7044. [DOI] [PubMed] [Google Scholar]
  25. Park S. K., Min D. S., Exton J. H. Definition of the protein kinase C interaction site of phospholipase D. Biochem Biophys Res Commun. 1998 Mar 17;244(2):364–367. doi: 10.1006/bbrc.1998.8275. [DOI] [PubMed] [Google Scholar]
  26. Park S. K., Provost J. J., Bae C. D., Ho W. T., Exton J. H. Cloning and characterization of phospholipase D from rat brain. J Biol Chem. 1997 Nov 14;272(46):29263–29271. doi: 10.1074/jbc.272.46.29263. [DOI] [PubMed] [Google Scholar]
  27. Reid T., Furuyashiki T., Ishizaki T., Watanabe G., Watanabe N., Fujisawa K., Morii N., Madaule P., Narumiya S. Rhotekin, a new putative target for Rho bearing homology to a serine/threonine kinase, PKN, and rhophilin in the rho-binding domain. J Biol Chem. 1996 Jun 7;271(23):13556–13560. doi: 10.1074/jbc.271.23.13556. [DOI] [PubMed] [Google Scholar]
  28. Sahai E., Alberts A. S., Treisman R. RhoA effector mutants reveal distinct effector pathways for cytoskeletal reorganization, SRF activation and transformation. EMBO J. 1998 Mar 2;17(5):1350–1361. doi: 10.1093/emboj/17.5.1350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Self A. J., Paterson H. F., Hall A. Different structural organization of Ras and Rho effector domains. Oncogene. 1993 Mar;8(3):655–661. [PubMed] [Google Scholar]
  30. Singer W. D., Brown H. A., Jiang X., Sternweis P. C. Regulation of phospholipase D by protein kinase C is synergistic with ADP-ribosylation factor and independent of protein kinase activity. J Biol Chem. 1996 Feb 23;271(8):4504–4510. doi: 10.1074/jbc.271.8.4504. [DOI] [PubMed] [Google Scholar]
  31. Sung T. C., Altshuller Y. M., Morris A. J., Frohman M. A. Molecular analysis of mammalian phospholipase D2. J Biol Chem. 1999 Jan 1;274(1):494–502. doi: 10.1074/jbc.274.1.494. [DOI] [PubMed] [Google Scholar]
  32. Sung T. C., Roper R. L., Zhang Y., Rudge S. A., Temel R., Hammond S. M., Morris A. J., Moss B., Engebrecht J., Frohman M. A. Mutagenesis of phospholipase D defines a superfamily including a trans-Golgi viral protein required for poxvirus pathogenicity. EMBO J. 1997 Aug 1;16(15):4519–4530. doi: 10.1093/emboj/16.15.4519. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Sung T. C., Zhang Y., Morris A. J., Frohman M. A. Structural analysis of human phospholipase D1. J Biol Chem. 1999 Feb 5;274(6):3659–3666. doi: 10.1074/jbc.274.6.3659. [DOI] [PubMed] [Google Scholar]
  34. Walker S. J., Wu W. J., Cerione R. A., Brown H. A. Activation of phospholipase D1 by Cdc42 requires the Rho insert region. J Biol Chem. 2000 May 26;275(21):15665–15668. doi: 10.1074/jbc.M000076200. [DOI] [PubMed] [Google Scholar]
  35. Xie Z., Ho W. T., Exton J. H. Association of N- and C-terminal domains of phospholipase D is required for catalytic activity. J Biol Chem. 1998 Dec 25;273(52):34679–34682. doi: 10.1074/jbc.273.52.34679. [DOI] [PubMed] [Google Scholar]
  36. Xie Z., Ho W. T., Exton J. H. Association of the N- and C-terminal domains of phospholipase D. Contribution of the conserved HKD motifs to the interaction and the requirement of the association for Ser/Thr phosphorylation of the enzyme. J Biol Chem. 2000 Aug 11;275(32):24962–24969. doi: 10.1074/jbc.M909745199. [DOI] [PubMed] [Google Scholar]
  37. Xie Z., Ho W. T., Exton J. H. Conserved amino acids at the C-terminus of rat phospholipase D1 are essential for enzymatic activity. Eur J Biochem. 2000 Dec;267(24):7138–7146. doi: 10.1046/j.1432-1327.2000.01816.x. [DOI] [PubMed] [Google Scholar]
  38. Yamazaki M., Zhang Y., Watanabe H., Yokozeki T., Ohno S., Kaibuchi K., Shibata H., Mukai H., Ono Y., Frohman M. A. Interaction of the small G protein RhoA with the C terminus of human phospholipase D1. J Biol Chem. 1999 Mar 5;274(10):6035–6038. doi: 10.1074/jbc.274.10.6035. [DOI] [PubMed] [Google Scholar]
  39. Zohar M., Teramoto H., Katz B. Z., Yamada K. M., Gutkind J. S. Effector domain mutants of Rho dissociate cytoskeletal changes from nuclear signaling and cellular transformation. Oncogene. 1998 Aug 27;17(8):991–998. doi: 10.1038/sj.onc.1202022. [DOI] [PubMed] [Google Scholar]
  40. Zong H., Raman N., Mickelson-Young L. A., Atkinson S. J., Quilliam L. A. Loop 6 of RhoA confers specificity for effector binding, stress fiber formation, and cellular transformation. J Biol Chem. 1999 Feb 19;274(8):4551–4560. doi: 10.1074/jbc.274.8.4551. [DOI] [PubMed] [Google Scholar]