Gene, 120 (1992) 325-326 0 1992 Elsevier Science Publishers B.V. All rights reserved. 0378-1119/92/$OS,OO
325
GENE 06733
Sequence of a canine cDNA protein *
clone encoding a Ran/TC4
GTP-binding
(MDCK II cells; Cunis familiaris; cDNA library; Ras-like proteins; canine Ran; canine TC4; cell cycle control)
Paul Dupree, Vesa M. Olkkonen and Philippe Chavrier** Cell Biology Programme, European Molecular Biology Laboratory, D-6900 Heidelberg, Germany
Received by J.K.C. Knowles: 24 April 1992; Accepted: 29 June 1992; Received at publishers: 14 July 1992
SUMMARY
We report the isolation and characterization of a canine cDNA encoding a 216~amino acid GTP-binding protein of the Ras superfamily. The protein is almost identical to the human TC4 [Drivas et al., Mol. Cell. Biol. 10 (1990) 1793-17981 and Ran [Bischoff and Ponstingl, Proc. Natl. Acad. Sci. USA 88 (1991) 10830-10834; Nature 354 (1991) 80-821 proteins, the latter of which has been found to be involved in cell cycle control. F~e~ore, the protein is highly similar to the fission yeast spil gene product [Matsumoto and Beach, Cell 66 (1991) 347-3601. The high degree of evolutionary conservation in this protein suggests that it plays a vital role in the eukaryotic cell.
The large effort currently invested in cloning and characterizing new Ras-related GTP-binding proteins is revealing the structural and functional versatility of this protein superfamily. Ras-like proteins have been shown to be involved in varied cellular processes, including signal transduction (for review, see Ridley and Hall, 1992), regulation of intrace~~~ transport (for review, see Pfeffer, 1992), cytoskeleton organization (Paterson et al., 1990), regulation of phagocyte oxygen radical production (Abo et al., 1991; Knaus et al., 1991), and, as recently demonstrated,
Correspondence to: Dr. P. Dupree, European Molecular Biology Laboratory, Postfach 10.2209, D-6900 Heidelberg, Germany. Tel. (49-6221)387336; Fax (49-6221)3873~. * On request, the authors will supply detailed experimental evidence for the conclusions reached in this Brief Note. ** Present address: Centre d’Immunologie de Marseille-Luminy, Case 906, 13288 Marseille Cedex 9, France. Tel. (33)91269466.
Abbreviations: aa, amino acid(s); bp, base pair(s), cDNA, DNA complementary to RNA; kb, kilobase or 1000 bp; MDCK, Madm-Darby canine kidney; nt, nucleotide(s); ORF, open reading frame; PCR, polymerase chain reaction.
control of the cell cycle (Bischoff and Ponstin& 1991a,b; Matsumoto and Beach, 1991). A cDNA fragment containing the 5’ end of the mouse Ran/TC4 ORF (Chavrier et al., 1992) was used for probing an MDCKII cell cDNA library constructed in the Iz ZAP11 phage (Stratagene, La folla, CA). This mouse cDNA fragment had been o~~n~ly cloned using a degenerate o~god~xy~bonucleotide corresponding to a conserved sequence (aa WDTAGQE; underlined in Fig. 1) of Ras-like proteins, forming part of the GTP-binding pocket, and a 5’ RACE (rapid amplification of cDNA ends) PCR approach. Ten of 3 x lo5 screened plaques strongly hybridized with the 210-bp probe. All ten phages had inserts of similar size (I .Okb), probably representing copies of the same original cDNA molecule generated in the library-amplification step. The nt sequence of this cDNA was determined and analyzed using the GCG program set (Devereux et al., 1984). It was found to contain an ORF of 648 bp, corresponding to a protein of 216 aa and a deduced MT of 24423 (Fig. 1). The protein belongs to the superfamily of Ras-related GTP-boding proteins. It has no C-terminal Cys-cont~ning
326 tical to the human sequence. Furthermore, the protein predicted is 81.5 y0 identical to the fission yeast spil gene product identified as a suppressor of the piml (premature initiation of mitosis) mutation (Matsumoto and Beach, 1991). The high degree of evolutionary conservation of the Ran/TC4/Spil proteins is remarkable and, together with the functional and biochemical data available (Bischoff and Ponstingl, 1991a,b; Matsumoto and Beach, 1991), it indicates a central role for these Ras-related GTP-binding proteins in cell cycle control.
199 61
This work was supported in part by grants from the Royal Society (P.D.), European Molecular Biology Organisation (V.M.O.), and European Molecular Biology Laboratory (P.C.).
REFERENCES Abo, A., Pick, E., Hall, A., Totty, N., Teahan, Activation
of the NADPH Nature
protein p21”“.
USA 88 (1991a)
679
Bischoff,
Fig. and GTPsequence, Arg lz9, is boxed. The aa conserved in all Ras-superfamily binding proteins (Valencia et al., 1991) are underlined. The EMBL Data Library/GenBank
F.R.
change
accession
No. of the sequence
is Z11922.
H.: Mitotic regulator
ras-related
polypeptide.
protein RCCl
is com-
Proc. Natl. Acad.
Sci.
10830-10834.
and Ponstingl,
H.: Catalysis
on Ran by the mitotic regulator
of guanine RCCl.
nucleotide
Nature
ex-
354 (1991b)
80-82. Chavrier,
P., Simons,
K. and Zerial, M.: The complexity
Rho GTP-binding
subfamilies
of the Rab and
revealed by a PCR cloning approach.
Gene 112 (1992) 261-264. Devereux,
motif, which is the site of isoprenylation in other Ras-like proteins, nor an N-terminal myristylation signal found in the ARF family GTP-binding proteins. The post-translational modifications are required for the membraneassociation of these proteins (Pfeffer, 1992). The protein is nearly identical to the human teratocarcinoma cell line (Ntera2) gene product TC4 (Drivas et al., 1990; Bischoff and Ponstingl, 1991a), the only difference being replacement by an Arg129 (in the MDCK II sequence, boxed in the figure) of the Ser in the human sequence. The aa sequences determined of peptides from another human protein, Ran (Ras-related nuclear protein, which is possibly the same as TC4), found to be associated with the mitotic regulator RCCl in HeLa cells (Bischoff and Ponstingl, 1991a), are also identical to the predicted MDCK II sequence, with the same single aa substitution as indicated above. At the nt level the canine sequence is 87.6% iden-
C.G. and Segal, A.W.:
involves the small GTP-binding
353 (1991) 668-670.
Bischoff, F.R. and Ponstingl, plexed with a nuclear
oxidase
J., Haeberli,
P. and Smithies,
quence analysis programs
0.: A comprehensive
set of se-
for the VAX. Nucleic Acids Res. 12 (1984)
387-395. Drivas,
G.T., Shih, A., Coutavas,
Characterization teratocarcinoma Knaus,
U.G.,
G.M.:
Heyworth,
Regulation
GTP-binding Matsumoto, Paterson,
P.G.,
RCCl
Evans,
of phagocyte
protein
T., Cumutte,
oxygen
D.: Premature
or an interacting
H.F., Self, A.J., Garrett,
A.: Microinjection morphology.
P.:
radical
J.T. and Bokoch, production
by the
Rat 2. Science 254 (1991) 1512-1515.
T. and Beach,
lacking
E., Rush, M.G. and d’Eustachio,
of four novel ras-like genes expressed in a human cell line. Mol. Cell. Biol. 10 (1990) 1793-1798.
GTPase.
initiation
of mitosis in yeast
Cell 66 (1991) 347-360.
M.D., Just, I., Aktories,
of recombinant
K. and Hall,
p2 1rh0 induces rapid changes in cell
J. Cell. Biol. 111 (1990) 1001-1007.
Pfeffer, S.R.: GTP-binding
proteins in intracellular
transport.
Trends Cell
Biol. 2 (1992) 41-46. Ridley, A.J. and Hall, A.: Function 497-49s. Valencia,
A., Chardin,
P., Wittinghofer,
tein family: evolutionary chemistry
for Ras in sight. Nature A. and Sander,
tree and role of conserved
30 (1991) 4637-4648.
355 (1992)
C.: The ras proamino acids. Bio-
325
GENE 06733
Sequence of a canine cDNA protein *
clone encoding a Ran/TC4
GTP-binding
(MDCK II cells; Cunis familiaris; cDNA library; Ras-like proteins; canine Ran; canine TC4; cell cycle control)
Paul Dupree, Vesa M. Olkkonen and Philippe Chavrier** Cell Biology Programme, European Molecular Biology Laboratory, D-6900 Heidelberg, Germany
Received by J.K.C. Knowles: 24 April 1992; Accepted: 29 June 1992; Received at publishers: 14 July 1992
SUMMARY
We report the isolation and characterization of a canine cDNA encoding a 216~amino acid GTP-binding protein of the Ras superfamily. The protein is almost identical to the human TC4 [Drivas et al., Mol. Cell. Biol. 10 (1990) 1793-17981 and Ran [Bischoff and Ponstingl, Proc. Natl. Acad. Sci. USA 88 (1991) 10830-10834; Nature 354 (1991) 80-821 proteins, the latter of which has been found to be involved in cell cycle control. F~e~ore, the protein is highly similar to the fission yeast spil gene product [Matsumoto and Beach, Cell 66 (1991) 347-3601. The high degree of evolutionary conservation in this protein suggests that it plays a vital role in the eukaryotic cell.
The large effort currently invested in cloning and characterizing new Ras-related GTP-binding proteins is revealing the structural and functional versatility of this protein superfamily. Ras-like proteins have been shown to be involved in varied cellular processes, including signal transduction (for review, see Ridley and Hall, 1992), regulation of intrace~~~ transport (for review, see Pfeffer, 1992), cytoskeleton organization (Paterson et al., 1990), regulation of phagocyte oxygen radical production (Abo et al., 1991; Knaus et al., 1991), and, as recently demonstrated,
Correspondence to: Dr. P. Dupree, European Molecular Biology Laboratory, Postfach 10.2209, D-6900 Heidelberg, Germany. Tel. (49-6221)387336; Fax (49-6221)3873~. * On request, the authors will supply detailed experimental evidence for the conclusions reached in this Brief Note. ** Present address: Centre d’Immunologie de Marseille-Luminy, Case 906, 13288 Marseille Cedex 9, France. Tel. (33)91269466.
Abbreviations: aa, amino acid(s); bp, base pair(s), cDNA, DNA complementary to RNA; kb, kilobase or 1000 bp; MDCK, Madm-Darby canine kidney; nt, nucleotide(s); ORF, open reading frame; PCR, polymerase chain reaction.
control of the cell cycle (Bischoff and Ponstin& 1991a,b; Matsumoto and Beach, 1991). A cDNA fragment containing the 5’ end of the mouse Ran/TC4 ORF (Chavrier et al., 1992) was used for probing an MDCKII cell cDNA library constructed in the Iz ZAP11 phage (Stratagene, La folla, CA). This mouse cDNA fragment had been o~~n~ly cloned using a degenerate o~god~xy~bonucleotide corresponding to a conserved sequence (aa WDTAGQE; underlined in Fig. 1) of Ras-like proteins, forming part of the GTP-binding pocket, and a 5’ RACE (rapid amplification of cDNA ends) PCR approach. Ten of 3 x lo5 screened plaques strongly hybridized with the 210-bp probe. All ten phages had inserts of similar size (I .Okb), probably representing copies of the same original cDNA molecule generated in the library-amplification step. The nt sequence of this cDNA was determined and analyzed using the GCG program set (Devereux et al., 1984). It was found to contain an ORF of 648 bp, corresponding to a protein of 216 aa and a deduced MT of 24423 (Fig. 1). The protein belongs to the superfamily of Ras-related GTP-boding proteins. It has no C-terminal Cys-cont~ning
326 tical to the human sequence. Furthermore, the protein predicted is 81.5 y0 identical to the fission yeast spil gene product identified as a suppressor of the piml (premature initiation of mitosis) mutation (Matsumoto and Beach, 1991). The high degree of evolutionary conservation of the Ran/TC4/Spil proteins is remarkable and, together with the functional and biochemical data available (Bischoff and Ponstingl, 1991a,b; Matsumoto and Beach, 1991), it indicates a central role for these Ras-related GTP-binding proteins in cell cycle control.
199 61
This work was supported in part by grants from the Royal Society (P.D.), European Molecular Biology Organisation (V.M.O.), and European Molecular Biology Laboratory (P.C.).
REFERENCES Abo, A., Pick, E., Hall, A., Totty, N., Teahan, Activation
of the NADPH Nature
protein p21”“.
USA 88 (1991a)
679
Bischoff,
Fig. and GTPsequence, Arg lz9, is boxed. The aa conserved in all Ras-superfamily binding proteins (Valencia et al., 1991) are underlined. The EMBL Data Library/GenBank
F.R.
change
accession
No. of the sequence
is Z11922.
H.: Mitotic regulator
ras-related
polypeptide.
protein RCCl
is com-
Proc. Natl. Acad.
Sci.
10830-10834.
and Ponstingl,
H.: Catalysis
on Ran by the mitotic regulator
of guanine RCCl.
nucleotide
Nature
ex-
354 (1991b)
80-82. Chavrier,
P., Simons,
K. and Zerial, M.: The complexity
Rho GTP-binding
subfamilies
of the Rab and
revealed by a PCR cloning approach.
Gene 112 (1992) 261-264. Devereux,
motif, which is the site of isoprenylation in other Ras-like proteins, nor an N-terminal myristylation signal found in the ARF family GTP-binding proteins. The post-translational modifications are required for the membraneassociation of these proteins (Pfeffer, 1992). The protein is nearly identical to the human teratocarcinoma cell line (Ntera2) gene product TC4 (Drivas et al., 1990; Bischoff and Ponstingl, 1991a), the only difference being replacement by an Arg129 (in the MDCK II sequence, boxed in the figure) of the Ser in the human sequence. The aa sequences determined of peptides from another human protein, Ran (Ras-related nuclear protein, which is possibly the same as TC4), found to be associated with the mitotic regulator RCCl in HeLa cells (Bischoff and Ponstingl, 1991a), are also identical to the predicted MDCK II sequence, with the same single aa substitution as indicated above. At the nt level the canine sequence is 87.6% iden-
C.G. and Segal, A.W.:
involves the small GTP-binding
353 (1991) 668-670.
Bischoff, F.R. and Ponstingl, plexed with a nuclear
oxidase
J., Haeberli,
P. and Smithies,
quence analysis programs
0.: A comprehensive
set of se-
for the VAX. Nucleic Acids Res. 12 (1984)
387-395. Drivas,
G.T., Shih, A., Coutavas,
Characterization teratocarcinoma Knaus,
U.G.,
G.M.:
Heyworth,
Regulation
GTP-binding Matsumoto, Paterson,
P.G.,
RCCl
Evans,
of phagocyte
protein
T., Cumutte,
oxygen
D.: Premature
or an interacting
H.F., Self, A.J., Garrett,
A.: Microinjection morphology.
P.:
radical
J.T. and Bokoch, production
by the
Rat 2. Science 254 (1991) 1512-1515.
T. and Beach,
lacking
E., Rush, M.G. and d’Eustachio,
of four novel ras-like genes expressed in a human cell line. Mol. Cell. Biol. 10 (1990) 1793-1798.
GTPase.
initiation
of mitosis in yeast
Cell 66 (1991) 347-360.
M.D., Just, I., Aktories,
of recombinant
K. and Hall,
p2 1rh0 induces rapid changes in cell
J. Cell. Biol. 111 (1990) 1001-1007.
Pfeffer, S.R.: GTP-binding
proteins in intracellular
transport.
Trends Cell
Biol. 2 (1992) 41-46. Ridley, A.J. and Hall, A.: Function 497-49s. Valencia,
A., Chardin,
P., Wittinghofer,
tein family: evolutionary chemistry
for Ras in sight. Nature A. and Sander,
tree and role of conserved
30 (1991) 4637-4648.
355 (1992)
C.: The ras proamino acids. Bio-
