Identification and Characterization of Rain, a Novel Ras-interacting Protein with a Unique Subcellular Localization

Abstract

The Ras small GTPase functions as a signaling node and is activated by extracellular stimuli. Upon activation, Ras interacts with a spectrum of functionally diverse downstream effectors and stimulates multiple cytoplasmic signaling cascades that regulate cellular proliferation, differentiation, and apoptosis. In addition to the association of Ras with the plasma membrane, recent studies have established an association of Ras with Golgi membranes. Whereas the effectors of signal transduction by activated, plasma membrane-localized Ras are well characterized, very little is known about the effectors used by Golgi-localized Ras. In this study, we report the identification of a novel Ras-interacting protein, Rain, that may serve as an effector for endomembrane-associated Ras. Rain does not share significant sequence similarity with any known mammalian proteins, but contains a Ras-associating domain that is found in RalGDS, AF-6, and other characterized Ras effectors. Rain interacts with Ras in a GTP-dependent manner in vitro and in vivo, requires an intact Ras core effector-binding domain for this interaction, and thus fits the definition of a Ras effector. Unlike other Ras effectors, however, Rain is localized to perinuclear, juxta-Golgi vesicles in intact cells and is recruited to the Golgi by activated Ras. Finally, we found that Rain cooperates with activated Raf and causes synergistic transformation of NIH3T3 cells. Taken together, these observations support a role for Rain as a novel protein that can serve as an effector of endomembrane-localized Ras. The Ras small GTPase functions as a signaling node and is activated by extracellular stimuli. Upon activation, Ras interacts with a spectrum of functionally diverse downstream effectors and stimulates multiple cytoplasmic signaling cascades that regulate cellular proliferation, differentiation, and apoptosis. In addition to the association of Ras with the plasma membrane, recent studies have established an association of Ras with Golgi membranes. Whereas the effectors of signal transduction by activated, plasma membrane-localized Ras are well characterized, very little is known about the effectors used by Golgi-localized Ras. In this study, we report the identification of a novel Ras-interacting protein, Rain, that may serve as an effector for endomembrane-associated Ras. Rain does not share significant sequence similarity with any known mammalian proteins, but contains a Ras-associating domain that is found in RalGDS, AF-6, and other characterized Ras effectors. Rain interacts with Ras in a GTP-dependent manner in vitro and in vivo, requires an intact Ras core effector-binding domain for this interaction, and thus fits the definition of a Ras effector. Unlike other Ras effectors, however, Rain is localized to perinuclear, juxta-Golgi vesicles in intact cells and is recruited to the Golgi by activated Ras. Finally, we found that Rain cooperates with activated Raf and causes synergistic transformation of NIH3T3 cells. Taken together, these observations support a role for Rain as a novel protein that can serve as an effector of endomembrane-localized Ras. Ras proteins (H-, N- and K-Ras) are essential components of the extracellular signaling cascades that control cellular proliferation, differentiation, and apoptosis. Mutated alleles of Ras are found in a variety of human tumors (1Bos J.L. Cancer Res. 1989; 49: 4682-4689PubMed Google Scholar). Oncogenic Ras induces the growth and morphological transformation of many cell types in vitro and is directly linked to tumorigenesis in animal models (2Barbacid M. Annu. Rev. Biochem. 1987; 56: 779-827Crossref PubMed Scopus (3792) Google Scholar). Ras is a plasma membrane-localized protein that cycles between GDP-bound (inactive) and GTP-bound (active) states. After activation by external stimuli, Ras-GTP binds with multiple downstream effectors through the core Ras effector-binding domain (residues 32–40), which results in the stimulation of signaling cascades that regulate cytoplasmic and nuclear events. A growing number of Ras effectors have been identified over the years, with the Raf serine/threonine kinases, the phosphatidylinositol 3-kinase (PI3K) 1The abbreviations used are: PI3K, phosphatidylinositol 3-kinase; RA, Ras-associating; DB, binding domain; GFP, green fluorescent protein; CFP, cyan fluorescent protein; HEK, human embryonic kidney; HA, hemagglutinin; GST, glutathione S-transferase; RBD, Ras-binding domain; RA, Ras-associated; DIL, dilute; YFP, yellow fluorescent protein; GalT, galactosyl transferase. lipid kinases, and the Ral guanine nucleotide exchange factors among the best characterized (3Downward J. Curr. Opin. Genet. Dev. 1998; 8: 49-54Crossref PubMed Scopus (514) Google Scholar, 4Marshall C.J. Curr. Opin. Cell Biol. 1996; 8: 197-204Crossref PubMed Scopus (475) Google Scholar, 5Shields J.M. Pruitt K. McFall A. Shaub A. Der C.J. Trends Cell Biol. 2000; 10: 147-154Abstract Full Text Full Text PDF PubMed Scopus (692) Google Scholar, 6Wolthuis R.M. Bos J.L. Curr. Opin. Genet. Dev. 1999; 9: 112-117Crossref PubMed Scopus (128) Google Scholar). Ras activation of effector function is mediated, in part, by the recruitment of these cytoplasmic proteins to the plasma membrane. Recent studies involving live-cell imaging, electron microscopy, and fluorescence resonance energy transfer have shown that in addition to the plasma membrane, H-Ras and N-Ras, but not K-Ras, localize to intracellular membranes of the endoplasmic reticulum and Golgi (7Choy E. Chiu V.K. Silletti J. Feoktistov M. Morimoto T. Michaelson D. Ivanov I.E. Philips M.R. Cell. 1999; 98: 69-80Abstract Full Text Full Text PDF PubMed Scopus (628) Google Scholar). Philips and colleagues (8Bivona T.G. Perez de Castro I. Ahearn I.M. Grana T.M. Chiu V.K. Lockyer P.J. Cullen P.J. Pellicer A. Cox A.D. Philips M.R. Nature. 2003; 424: 694-698Crossref PubMed Scopus (361) Google Scholar) have shown that Golgi-localized Ras is activated through a Src/phospholipase Cγ1/Ras guanyl nucleotide-releasing protein pathway that is distinct from the classic protein receptor tyrosine kinase/Grb2/SOS pathway that activates Ras at the plasma membrane. Furthermore, they demonstrated that endomembrane-associated Ras is biologically active because silencing of endogenous Ras guanyl nucleotide-releasing protein-mediated activation of Ras completely blocks T-cell receptor-stimulated Ras activation in Jurkat cells and neuronal differentiation in PC-12 cells. In NIH3T3 transformation assays, a constitutively active, palmitoylation-deficient H-Ras mutant that is localized and activated on endomembranes exhibits transforming activity comparable with that of wild-type activated H-Ras (9Chiu V.K. Bivona T. Hach A. Sajous J.B. Silletti J. Wiener H. Johnson II, R.L. Cox A.D. Philips M.R. Nat. Cell Biol. 2002; 4: 343-350Crossref PubMed Scopus (522) Google Scholar). Whether the endomembrane and plasma membrane pools of biologically active Ras use the same set of effector molecules remains unknown. Genetically engineered variants of Raf, PI3K, and Ral guanine nucleotide exchange factors that are targeted to the plasma membrane in the absence of Ras activation are constitutively active as signaling molecules (10Leevers S.J. Paterson H.F. Marshall C.J. Nature. 1994; 369: 411-414Crossref PubMed Scopus (890) Google Scholar, 11Stokoe D. Macdonald S.G. Cadwallader K. Symons M. Hancock J.F. Science. 1994; 264: 1463-1467Crossref PubMed Scopus (851) Google Scholar, 12Rodriguez-Viciana P. Warne P.H. Khwaja A. Marte B.M. Pappin D. Das P. Waterfield M.D. Ridley A. Downward J. Cell. 1997; 89: 457-467Abstract Full Text Full Text PDF PubMed Scopus (965) Google Scholar, 13Wolthuis R.M. de Ruiter N.D. Cool R.H. Bos J.L. EMBO J. 1997; 16: 6748-6761Crossref PubMed Scopus (145) Google Scholar), establishing these proteins as bona fide effectors of plasma membrane-localized Ras. The identification of the effectors of endomembrane-associated Ras is the next challenge in understanding the role and importance of Ras signaling originating from endomembranes. Herein, we report the identification of Rain, a novel Ras-interacting protein that displays the characteristics of an effector of endomembrane-localized Ras. First, Rain possesses a Ras-associating (RA) domain homologous to the RA domains of other Ras effectors, including the Ral guanine nucleotide exchange factors, AF-6, Rin1, and phospholipase Cϵ (14Ponting C.P. Benjamin D.R. Trends Biochem. Sci. 1996; 21: 422-425Abstract Full Text PDF PubMed Scopus (180) Google Scholar). Second, like Raf and all other known Ras effectors, Rain preferentially binds to the GTP-loaded form of Ras in vitro and in vivo, and the interaction of Rain with Ras is abolished by mutations in the core effector domain of Ras. Third, we show that Rain localizes to a perinuclear, juxta-Golgi region in intact cells and is recruited to the Golgi by active Ras. Finally, we determined that co-expression of Rain together with activated Raf causes synergistic transformation of NIH3T3 cells. These data support a role for Rain as a novel effector for the activated pool of Ras localized to endomembranes in mammalian cells. Yeast Two-hybrid Assays—Individual Gal4 DNA binding domain (DB)-Ras fusion constructs (DB-Ras G12V,C186S; DB-Ras G12V,T35S,C186S; DB-Ras G12V,E37G,C186S; and DB-Ras G12V,Y40C,C186S) were generated by standard procedures. Full-length cDNA sequences encoding effector domain mutants of activated H-Ras G12V were amplified by PCR using primers to introduce 5′-BamHI and 3′-EcoRI sites and to the sequence to a of the of the H-Ras signaling The PCR were the were in using as the and a human cDNA were on and were for activity as in Scholar). The from and in were and characterized as in the A Gal4 in generated using between Rain and Ras were by of from A. with Rain and small in as fusion proteins from A. and M. PubMed Scopus Google Scholar). were and for activity as of the Full-length Rain from the by and sequence from the and used to the cDNA sequence encoding the of A of by and the Rain Rain with and the the of Rain A with a of the with to a encoding the Rain and this Rain with to a Rain cDNA the HA, and mammalian encoding Rain at the with green fluorescent protein Rain generated by PCR of the sequence and The same used to an encoding cyan fluorescent Rain The human Rain cDNA sequence been the data number Cell and were in with and cells were in and cells were in with and NIH3T3 cells cells and cells were using the standard DNA cells were in and and cell were used for A a human Rain used to endogenous and Rain proteins in cell Rain with an NIH3T3 a to the of Rain to transformation through Ras signaling NIH3T3 cells were at cells and the next using and by the DNA constructs used for were M. Der C.J. Cell. Biol. 1996; 16: PubMed Scopus Google Scholar), P.J. Der C.J. Cell 2002; Google Scholar), and Rain is an constitutively activated of human is a constitutively activated mutant of human were growth and The of for and using are as number of from from the were and with Rain Rain in the we used from using and The primers for the Rain cDNA were as and were to a at the of the Rain of from were using and for and of amplified by PCR with for human Rain a human with a cDNA of Rain were at in for and to Rain protein from an were by in were by and protein determined by of were by to membrane, and using the between Rain and glutathione fusion proteins the Ras-binding domain of and a region of Rain the RA domain were and from E. and H-Ras protein with by for at in of and were on the to and the to with binding and with and of the nucleotide with of proteins for at to glutathione were with binding in and by The of H-Ras protein to by using a the of Rain to to other small cells were with of DNA of activated of cells were in and and of cell were for at with of GST, proteins on glutathione were with by to membrane, and using an cells together with were to and in and of protein in a of were at with of to were with from the with and by proteins were by using and cells were at cells and with of of constructs using cells were with and using an as PubMed Scopus Google Scholar). using an fluorescence with a Rain at the cells were number on the and as with of and of from M. in the absence of of cells were in using a of a Ras-interacting Ras effectors, a using a G12V fusion protein as the and an human cDNA as the of were that interaction growth on and of The of the interaction with Ras G12V by with a variety of control not of the that cDNA sequences for the known Ras effectors, Rin1, RalGDS, and The encoding the same of these were and an of a and a A of the nucleotide data with the sequence of a significant with any known but identified sequence to of the sequence number that used to the cDNA by in the and to an The cDNA is to a number and contains an for a with a of the protein by this cDNA binds to we this protein Rain, for Ras-interacting Rain contains a RA domain (14Ponting C.P. Benjamin D.R. Trends Biochem. Sci. 1996; 21: 422-425Abstract Full Text PDF PubMed Scopus (180) Google Scholar) in the domain the to the RA domains of human a protein identified as a of a in human H. T. H. K. K. Res. Google Scholar) and as a effector Biol. 1994; PubMed Scopus Google Scholar). sequence of the RA domain of Rain with the RA domains of other known Ras-interacting proteins is shown in Rain possesses the found to for the interaction of other effectors with Ras in and in Sci. A. 1994; PubMed Scopus Google Scholar, M. K. A. 1999; PubMed Scopus Google In addition to the RA Rain contains a region that is to the domain binding found in many intracellular Rain contains a domain found in the of the proteins C.P. Trends Biochem. Sci. Full Text PDF PubMed Scopus Google Scholar). In the domain may in binding to R.L. J. Cell Biol. 1999; PubMed Scopus Google Scholar, K. J. Cell Biol. 1998; PubMed Scopus Google Scholar). is of the is the protein found to a the role of this domain in function is unknown. the sequence of Rain used to the protein data we identified a protein sequence that is to a of Rain number In this identified a human and protein number and that the same domain as These proteins, with the and are to a of proteins with a of protein domains Rain have been identified in of the human sequence data identified the Rain and The and the protein they to of the Rain cDNA with sequence that the Rain of and of which the region of the human a that the of the Rain cDNA in all human with found in of from that the Rain is active in the of with the of Rain in the of the Rain protein, we used a from human Rain to a of protein from with the of and in and a in the of the Rain were with protein from from NIH3T3 cells The the protein in the cells the same were using and were in of the with the Rain used to the completely abolished of the protein and on the of the proteins of were used to the same not together with the of Rain, these that the to endogenous Rain Rain to Ras in a GTP-dependent effectors preferentially to the activated form of Ras through a region to as the core effector domain Yeast were used to Rain interaction with Ras effector domain These results show that an intact Ras effector domain is for interaction with Rain a interaction with H-Ras effector domain interaction using the variants next determined the interaction is by the activation of Ras. Ras protein with and with a fusion protein the RA domain of Rain The of used as a The of Ras protein determined by using an shown in Rain a for Ras-GTP a binding for the we the of Ras to to Rain the RA were not that the RA domain is and for interaction with Ras. Taken together with the these results show that Rain the of an effector for Ras. next the interaction in mammalian cells. these NIH3T3 cells were with an for activated H-Ras and a control a from cell using an and the protein were by and using an that the Ras protein is in from cells These results that interaction in mammalian cells and a role for Rain as a effector of Ras. Rain to Ras many of the Ras effectors characterized to Raf, RalGDS, AF-6, with other proteins in addition to we used to the of the Rain RA domain to to other of the Ras of In with studies with the Gal4 DB-Ras G12V Rain with the G12V fusion protein Rain with and with activated and interaction between Rain and a constitutively active form of these we the of the Rain RA domain to to Ras proteins in mammalian cells. active of and were in cells and cell were with with and as In with with the activated and proteins and with B.M. A. Science. PubMed Scopus Google Scholar, J. J.M. E. S.J. Marshall J. Nature. PubMed Scopus Google Scholar, M. Biochem. J. 1994; PubMed Scopus Google Scholar, Castro Der C.J. J. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). interaction with activated to a not with and interaction between the control protein and any of the These results that the RA domain of Rain interacts with Ras and proteins and that Rain may not an effector for signaling in cells. Rain with of Ras effectors characterized to are and are recruited to plasma membrane Ras the of Rain, we cells with an encoding together with a plasma membrane association and to in a region of the cell activated the protein localized to the plasma membrane as well as to a region is that with we a pool of Ras with to a region the cells and fluorescence at the plasma membrane. The of of Ras been identified as Golgi (7Choy E. Chiu V.K. Silletti J. Feoktistov M. Morimoto T. Michaelson D. Ivanov I.E. Philips M.R. Cell. 1999; 98: 69-80Abstract Full Text Full Text PDF PubMed Scopus (628) Google Scholar, V.K. Bivona T. Hach A. Sajous J.B. Silletti J. Wiener H. Johnson II, R.L. Cox A.D. Philips M.R. Nat. Cell Biol. 2002; 4: 343-350Crossref PubMed Scopus (522) Google Scholar). that the of Rain and Ras the we and a in the absence of H-Ras shown in in the absence of activated in perinuclear, and cytoplasmic vesicles and not with in the of wild-type vesicles were found at the Golgi and the fluorescent signal with In cells a constitutively active mutant of association at the Golgi and we an of and In perinuclear, juxta-Golgi and not recruited to the Golgi with GDP-bound Ras These data that Rain is in juxta-Golgi vesicles in intact cells and is recruited to Golgi membranes in the of active Ras. in that Rain is an effector for activated Ras at the but not at the plasma membrane. Rain with Raf to transforming activity of Ras is a by the activation of signaling (3Downward J. Curr. Opin. Genet. Dev. 1998; 8: 49-54Crossref PubMed Scopus (514) Google Scholar, 4Marshall C.J. Curr. Opin. Cell Biol. 1996; 8: 197-204Crossref PubMed Scopus (475) Google Scholar, 5Shields J.M. Pruitt K. McFall A. Shaub A. Der C.J. Trends Cell Biol. 2000; 10: 147-154Abstract Full Text Full Text PDF PubMed Scopus (692) Google Scholar, 6Wolthuis R.M. Bos J.L. Curr. Opin. Genet. Dev. 1999; 9: 112-117Crossref PubMed Scopus (128) Google Scholar). for the importance of Ras effector proteins to Ras function is the use of A. A. M. Cell. Full Text PDF PubMed Scopus Google Scholar, Der C.J. Cell. Biol. PubMed Scopus Google Scholar, T. E. J. Biol. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). activated not transformation of NIH3T3 co-expression of these effectors with activated Raf results in a synergistic of transforming activity P. Warne P.H. Khwaja A. Marte B.M. Pappin D. Das P. Waterfield M.D. Ridley A. Downward J. Cell. 1997; 89: 457-467Abstract Full Text Full Text PDF PubMed Scopus (965) Google Scholar, T. E. J. Biol. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). the synergistic transforming activity of Rain, NIH3T3 cells were with an for the protein by the were not In NIH3T3 cells with an for activated Raf the of A and Rain and were in NIH3T3 a in of transformation comparable with that with and which are to in this Der C.J. Cell. Biol. PubMed Scopus Google Scholar). These data support the that Rain, activated in the of other Ras effectors, can Recent studies demonstrated in addition to the plasma membrane, H-Ras localizes to endomembranes of the endoplasmic reticulum and is activated in to growth stimulation (7Choy E. Chiu V.K. Silletti J. Feoktistov M. Morimoto T. Michaelson D. Ivanov I.E. Philips M.R. Cell. 1999; 98: 69-80Abstract Full Text Full Text PDF PubMed Scopus (628) Google Scholar, T.G. Perez de Castro I. Ahearn I.M. Grana T.M. Chiu V.K. Lockyer P.J. Cullen P.J. Pellicer A. Cox A.D. Philips M.R. Nature. 2003; 424: 694-698Crossref PubMed Scopus (361) Google Scholar, V.K. Bivona T. Hach A. Sajous J.B. Silletti J. Wiener H. Johnson II, R.L. Cox A.D. Philips M.R. Nat. Cell Biol. 2002; 4: 343-350Crossref PubMed Scopus (522) Google Scholar). is that signal from endomembrane-localized Ras is from plasma Ras (9Chiu V.K. Bivona T. Hach A. Sajous J.B. Silletti J. Wiener H. Johnson II, R.L. Cox A.D. Philips M.R. Nat. Cell Biol. 2002; 4: 343-350Crossref PubMed Scopus (522) Google in J.F. Nat. Rev. Cell. Biol. 2003; 4: PubMed Scopus Google Scholar), as to endomembrane-localized Ras in cells and which signaling of the Ras effectors identified to are and are recruited to the plasma membrane Ras that Golgi-localized Ras may effectors to of effectors a in understanding of endomembrane-localized Ras In the study, we report the identification of a Ras to all known effectors of Rain preferentially with activated and this interaction is on an intact Ras effector-binding in to Raf and other Ras effectors, Rain does not to a protein and does not to the plasma membrane Ras we found that Rain in a perinuclear, juxta-Golgi Ras activation, is recruited to the Golgi but not to the plasma membrane. we found that Rain cooperates with Raf in cellular transformation assays, that Rain may serve as an effector for Ras localized to activated endomembrane but not plasma membrane. identified Rain in a using constitutively active H-Ras as a to the established effectors of Rain contains an RA domain in the The RA domain of Rain binds preferentially to Ras-GTP through an intact Ras effector The sequence between the core effector domains of of the Ras of proteins Der C.J. Curr. Opin. Cell Biol. 2000; PubMed Scopus Google Scholar) the interaction of Ras effectors with other small Raf binds to and M. Biochem. J. 1994; PubMed Scopus Google Scholar), and binds to and T. EMBO J. 1996; PubMed Scopus Google Scholar, R.M. de I. Bos J.L. de J. J. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, M. Sci. A. 1994; PubMed Scopus Google Scholar, M. M. 1996; Google Scholar). In this study, we that the Rain RA domain interacts with and and a interaction with activated and of interaction is to that for T. M. A. J. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar), and recent demonstrated that these are functionally can serve as a of activation of K. Cell. Biol. 2003; PubMed Scopus Google Scholar) but functions as a effector of the P. M. 2003; Google Scholar) and as a of the activation cell M. M. M. K. J. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). have not the role of Rain in the that is found on endomembranes and is activated in that of cells to growth factors (9Chiu V.K. Bivona T. Hach A. Sajous J.B. Silletti J. Wiener H. Johnson II, R.L. Cox A.D. Philips M.R. Nat. Cell Biol. 2002; 4: 343-350Crossref PubMed Scopus (522) Google Scholar), is a that Rain functions as a effector of Ras and proteins in cells. with Ras effector domain variants determined that Rain binds to the but not to the mutants of activated H-Ras These Ras effector domain variants of binding to the effectors for Ras transformation A. A. M. Cell. Full Text PDF PubMed Scopus Google Scholar). binding to Raf but not to RalGDS, binds but not Raf PI3K, and binds but not Raf is to that is to NIH3T3 the transforming activity in human cells M. Der C.J. Cell. Biol. 1996; 16: PubMed Scopus Google Scholar, C.P. Sci. A. 1998; PubMed Scopus Google Scholar, Der C.J. Dev. 2002; 16: PubMed Scopus Google Scholar). found that co-expression of Rain Raf transforming which is with the effector domain interaction transformation of human cells to interaction with RalGDS, is that Rain in to the transforming with this effector domain mutant of Ras. is the that Rain localizes to a perinuclear, juxta-Golgi region and to the region of activated Ras. that Rain can serve as an effector of Golgi-localized Ras because cellular is by the activation of Ras. have this of Rain in a variety of mammalian cell using other of Rain protein HA, and In we were to the of by cell by the of Rain in and cells. Rain are in the of not endogenous Rain in by in cell by a at this we are to the of endogenous Rain in cells. are we to cell with in an to cell types that the of Rain in between the Rain and protein the RA domains to a to as a which is a of the of proteins C.P. Trends Biochem. Sci. Full Text PDF PubMed Scopus Google Scholar). In the domain is in the and may in binding to R.L. J. Cell Biol. 1999; PubMed Scopus Google Scholar, K. J. Cell Biol. 1998; PubMed Scopus Google Scholar). and colleagues M. K. Morimoto K. A. M. J. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, H. J. K. H. M. A. K. H. H. A. J. Cell Biol. 1999; PubMed Scopus Google Scholar) have that the of AF-6, localizes to the Golgi in addition to at data that the and the of Rain are for to a perinuclear, juxta-Golgi region not is to that Rain binds to Ras-GTP through and of Ras to the plasma membrane with vesicles through In we have identified Rain as a effector of and function in mammalian cells. In to other Ras effectors, Rain does not with activated Ras at the plasma membrane. Rain may serve as an effector for endomembrane-associated Ras and a function for endomembrane-localized Ras that from the function of Ras at the plasma membrane. studies an of the of the interaction and role in cellular and and Philips for that were used in these