Gene, 0
118 (1992) 297-298 Elsevier Science Publishers
1992
GENE
B.V. All rights reserved.
297
0378-l 119/92/$05.00
06606
A mouse c-ros genomic clone: identification of a highly conserved 22-amino acid segment in the juxtamembrane domain* (Murine/human
Lakshmi
homology;
Narayana
Drosophila melanogaster sevenless gene; chicken)
protooncogene,
and Lalitha Nagarajan The Univrrsit.v of Texas M.D. Anderson Cancer Center, Houston, TX. USA
Depcrrtment of Hrmutology, Received
protein tyrosine kinase receptor;
by R. Padmanhaban:
22 November
1991; Accepted:
9 December
1991; Received
at publishers:
18 May 1992
SUMMARY
The mouse c-ros protooncogene genomic sequences have been cloned; an analysis of the partial genomic clone revealed a high conservation of the exons encoding the juxtamembrane (JM) and the 5’ most protein tyrosine kinase domains. We have identified a segment of 22 amino acids conserved between the human and mouse JM domains; this segment may have a critical role in the function of the c-ros-encoded protein tyrosine kinase receptor.
Protein tyrosine kinase receptors mediate signal transduction by binding to extracellar ligands. Typically these proteins consist of: (i) an N-terminal ligand-binding extracellular domain; (ii) a transmembrane domain consisting of a stretch of hydrophobic aa; (iii) a cytoplasmic JM segment; and (iv) a cytoplasmic PTK catalytic moiety (Ullrich and Schlessinger, 1990). The cellular homolog c-ros of the transforming sequences v-ros of avian myxosarcoma virus UR2 encodes a PTKR. An analysis of homologies among PTKRs reveals that the PTK domain of the human c-ROS protoncogene is closest to the homeotic gene, sevenless, of Drosophila melanogaster (Matsushime and Shibuya, 1990; Birchmeir et al., 1990; Chen et al., 1991). We cloned the mouse c-ros homolog in order to generate reagents to study its role in mammalian development. Characterization of a partial mouse c-ros genomic clone (Fig. 1) has identified a highly-conserved intron-exon structure between the mouse and human exons encoding the JM (exon 3) and the 5’-most of the exons encoding the PTK domain (exon 4).
TM human
+___‘Ipj
Fig. 1. Comparison
M.D. Anderson
Cancer Ctr., 1515 Holcombe
USA. Tel. (713) 792-8756; * On request, the authors the conclusions Abbreviations: kb, kilobase PTKR,
reached
of Hematology,
Blvd., Houston,
sushime
lines denote
introns.
c-ROS
vertical bars denote
Positions
HH
H
II
to the human
loci. For
nomenclature
(Mat-
exons. and horizontal
of JM (exon 3) and 5’-most
with reference
I
and mouse c-~0.7 genomic
we used the human
et al., 1986). Heavy
(exon 4) are indicated
I\
c-ROS
PTK exon phage clone.
The phROSHi6 human genomic probe (Nagarajan et al., 1986). used to screen the mouse genomic library, is shown in outlined letters. Plasmids pmros6.5,
pmros9.2
and pmros4.3 enzymes A, AwI;
are subclones
of the mouse c-ros phage
U and A cleave within mouse c-ros exons 3 B, BarnHI;
C, SacI; G, BglII: H, HindIII;
P, P.ytI; U, StuI; S, SalI; and X, Xhal.
TX 77030,
Fax (713) 794-5868. will provide detailed experimental
evidence for
in this Brief Note.
aa, amino acid(s); bp, base pair(s); JM, juxtamembrane; or 1000 bp; nt, nucleotide(s); PTK, protein tyrosine kinase;
PTK receptor:
of human c-ROS
the sake of consistency,
and 4, respectively. Box 24, Department
PTK
c- ROS clone
clone. Restriction Correspondence to: Dr. L. Nagarajan,
JM
ros (ROS), gene encoding
PTKR.
Homologies between the mouse, D. melanogaster, rat and human c-ros sequences are shown in Fig. 2. The overall homology between man and mouse at the nt level for exons 3 and 4 were 79% and 87”; (data not shown), and at the aa level, 75% and 92.6”/,, respectively (Fig. 2). A homol-
298 E3 JM .
1
Mouse:
VWHRSLKSHKASKKGLSVLNDNDKELAELRGLAA
Human:
~~~~R~~NR~SA~Q-VT~~INQ~~~~~~~~~~~~
Rat:
~~~~~~~N~~~T~R~~~~~~~~~Q~~~~~~~~~~
Chicken:
_--
QRW--
R-PAST-QI--VKE-----Q---M-E
:
:
:
D.
melanogaster:
:
+
L RAKK
:
- Q - R P S I W 8 N - - T -Q
Mouse:
E4 v GVGLANACYAVETVPTQEEIENLPAFPL
Human:
_________
Rat:
___-___-_
Chicken:
T
_
_
_
_
_
:
D.
melanogaster:
_
_
:
:
T Q Q QLM
]V
-[
KLSLR
_
:
STT-SD-D1
Mouse:
LLLGSGAFG
Human:
---------
____________________---
Rat:
_-_-_---_
____________________---
Chicken:
K______-_
-------L---AD----SR----
D.
RF-------
-
: melanogaster:
Fig. 2. Homology
- F
- NR
at the aa level between the human,
by the mouse c-ros exons 3 and 4. Colons
-
-
rat, mouse, chicken
_ -
[IQ
:
- KTEDSEEPQ[]R--I-
and D. melunogusfer
C-TOTexons 3 and 4. Dashes
denote related aa. and arc shown only for the D. melanoguster
sequence.
indicate
Plus denotes
identical
aa encoded
a 2-aa insertion
in the
D. nzela/wgcrsrer JM domain. Square brackets denote gaps introduced to obtain the optimum alignment. The conserved 22.aa segment is underlined. The invariant GXGXXG (doubly underlined) and the p-fluorosulfonyl-5’-benzoyladenosine (FSBA)-modifiable Lys (K*) are indicated. Arrowheads Indicate start of exons 3 (E3) and 4 (E4). Arrows M88609
and M88610,
indicate
start of the JM and PTK domains.
The Genbank
accession
Nos. for mouse c-w.7 cxons 3 and 4 are
respectively
ogy search between human, mouse and chicken at the aa level revealed the presence of a highly-conserved segment of 22 aa in the JM domain between mouse and human (100 y,, homology); the chicken sequences differ only in four aa within this segment (Fig. 2). The D. melanogaster sevenless sequences show patchy homology in this segment. In contrast, the entire kinase domain of D. melanogaster exhibits 70% homology to the rat and human c-ROS sequences (Matsushime and Shibuya, 1990; Birchmeir et al., 1990). Identification of the conserved 22-aa segment in the JM domain of c-ros is suggestive of a critical role for it in signal transduction and down regulation.
Birchmcicr, of ROSl
C., O’Neill, K., Riggs, M. and Wigler, M.: Characterization cDNA
from a human
glioblastoma
cell line. Proc.
Acad. Sci. USA 87 (1990) 4799-4803. Chen, J.. Hcllcr, D.. Peon, B., Kang, L. and Wang. oncogenc c-YO.Tcodes for a transmembrane sharing sequences and structural homology Drosophilu melunogusrer. Oncogenc Matsushime,
Natl.
L.H.: The proto-
tyrosine protein kinasc with .srve&s.c protein of
6 (199 1) 257-264.
H., Wang, L. and Shibuya,
M.: Human
c-ROSI
gene ho-
mologous to the v-rus sequence of UR2 sarcoma virus encodes a transmembrane receptorlike molecule. Mol. Cell. Biol. 6 (I 986) 30003004. Matsushimc, H. and Shibuya, M.: Tissue-specific expression of rat c-ro.\-I gene and partral structural similarity of its prcdictcd products with .wv protein of Drosophilcr melunogusrer. J. Virol. 64 (1990) 2 Il72125. Nagarajan.
L., Louie, E., Tsujimoto,
Y.. Baldurzi,
P.C., Huebner,
K. and
Croce, C.M.: The human c-ros gene (ROS) is located at chromosome REFERENCES
region 6q16-6q22. Proc. Natl. Acad. Sci. USA 83 (1986) 656X-6572. Ullrich, .4. and Schlessinger, J.: Signal transduction by rcccptors with
Basler, K. and Hafen, enless protein (1988) 299-311.
E.: Control
requires
of photoreceptor
a functional
tyrosine
cell fate by the sev-
kinase domain.
Cell 54
tyrosine
kinasc
activity.
Cell 61 (1990) 735-738.
118 (1992) 297-298 Elsevier Science Publishers
1992
GENE
B.V. All rights reserved.
297
0378-l 119/92/$05.00
06606
A mouse c-ros genomic clone: identification of a highly conserved 22-amino acid segment in the juxtamembrane domain* (Murine/human
Lakshmi
homology;
Narayana
Drosophila melanogaster sevenless gene; chicken)
protooncogene,
and Lalitha Nagarajan The Univrrsit.v of Texas M.D. Anderson Cancer Center, Houston, TX. USA
Depcrrtment of Hrmutology, Received
protein tyrosine kinase receptor;
by R. Padmanhaban:
22 November
1991; Accepted:
9 December
1991; Received
at publishers:
18 May 1992
SUMMARY
The mouse c-ros protooncogene genomic sequences have been cloned; an analysis of the partial genomic clone revealed a high conservation of the exons encoding the juxtamembrane (JM) and the 5’ most protein tyrosine kinase domains. We have identified a segment of 22 amino acids conserved between the human and mouse JM domains; this segment may have a critical role in the function of the c-ros-encoded protein tyrosine kinase receptor.
Protein tyrosine kinase receptors mediate signal transduction by binding to extracellar ligands. Typically these proteins consist of: (i) an N-terminal ligand-binding extracellular domain; (ii) a transmembrane domain consisting of a stretch of hydrophobic aa; (iii) a cytoplasmic JM segment; and (iv) a cytoplasmic PTK catalytic moiety (Ullrich and Schlessinger, 1990). The cellular homolog c-ros of the transforming sequences v-ros of avian myxosarcoma virus UR2 encodes a PTKR. An analysis of homologies among PTKRs reveals that the PTK domain of the human c-ROS protoncogene is closest to the homeotic gene, sevenless, of Drosophila melanogaster (Matsushime and Shibuya, 1990; Birchmeir et al., 1990; Chen et al., 1991). We cloned the mouse c-ros homolog in order to generate reagents to study its role in mammalian development. Characterization of a partial mouse c-ros genomic clone (Fig. 1) has identified a highly-conserved intron-exon structure between the mouse and human exons encoding the JM (exon 3) and the 5’-most of the exons encoding the PTK domain (exon 4).
TM human
+___‘Ipj
Fig. 1. Comparison
M.D. Anderson
Cancer Ctr., 1515 Holcombe
USA. Tel. (713) 792-8756; * On request, the authors the conclusions Abbreviations: kb, kilobase PTKR,
reached
of Hematology,
Blvd., Houston,
sushime
lines denote
introns.
c-ROS
vertical bars denote
Positions
HH
H
II
to the human
loci. For
nomenclature
(Mat-
exons. and horizontal
of JM (exon 3) and 5’-most
with reference
I
and mouse c-~0.7 genomic
we used the human
et al., 1986). Heavy
(exon 4) are indicated
I\
c-ROS
PTK exon phage clone.
The phROSHi6 human genomic probe (Nagarajan et al., 1986). used to screen the mouse genomic library, is shown in outlined letters. Plasmids pmros6.5,
pmros9.2
and pmros4.3 enzymes A, AwI;
are subclones
of the mouse c-ros phage
U and A cleave within mouse c-ros exons 3 B, BarnHI;
C, SacI; G, BglII: H, HindIII;
P, P.ytI; U, StuI; S, SalI; and X, Xhal.
TX 77030,
Fax (713) 794-5868. will provide detailed experimental
evidence for
in this Brief Note.
aa, amino acid(s); bp, base pair(s); JM, juxtamembrane; or 1000 bp; nt, nucleotide(s); PTK, protein tyrosine kinase;
PTK receptor:
of human c-ROS
the sake of consistency,
and 4, respectively. Box 24, Department
PTK
c- ROS clone
clone. Restriction Correspondence to: Dr. L. Nagarajan,
JM
ros (ROS), gene encoding
PTKR.
Homologies between the mouse, D. melanogaster, rat and human c-ros sequences are shown in Fig. 2. The overall homology between man and mouse at the nt level for exons 3 and 4 were 79% and 87”; (data not shown), and at the aa level, 75% and 92.6”/,, respectively (Fig. 2). A homol-
298 E3 JM .
1
Mouse:
VWHRSLKSHKASKKGLSVLNDNDKELAELRGLAA
Human:
~~~~R~~NR~SA~Q-VT~~INQ~~~~~~~~~~~~
Rat:
~~~~~~~N~~~T~R~~~~~~~~~Q~~~~~~~~~~
Chicken:
_--
QRW--
R-PAST-QI--VKE-----Q---M-E
:
:
:
D.
melanogaster:
:
+
L RAKK
:
- Q - R P S I W 8 N - - T -Q
Mouse:
E4 v GVGLANACYAVETVPTQEEIENLPAFPL
Human:
_________
Rat:
___-___-_
Chicken:
T
_
_
_
_
_
:
D.
melanogaster:
_
_
:
:
T Q Q QLM
]V
-[
KLSLR
_
:
STT-SD-D1
Mouse:
LLLGSGAFG
Human:
---------
____________________---
Rat:
_-_-_---_
____________________---
Chicken:
K______-_
-------L---AD----SR----
D.
RF-------
-
: melanogaster:
Fig. 2. Homology
- F
- NR
at the aa level between the human,
by the mouse c-ros exons 3 and 4. Colons
-
-
rat, mouse, chicken
_ -
[IQ
:
- KTEDSEEPQ[]R--I-
and D. melunogusfer
C-TOTexons 3 and 4. Dashes
denote related aa. and arc shown only for the D. melanoguster
sequence.
indicate
Plus denotes
identical
aa encoded
a 2-aa insertion
in the
D. nzela/wgcrsrer JM domain. Square brackets denote gaps introduced to obtain the optimum alignment. The conserved 22.aa segment is underlined. The invariant GXGXXG (doubly underlined) and the p-fluorosulfonyl-5’-benzoyladenosine (FSBA)-modifiable Lys (K*) are indicated. Arrowheads Indicate start of exons 3 (E3) and 4 (E4). Arrows M88609
and M88610,
indicate
start of the JM and PTK domains.
The Genbank
accession
Nos. for mouse c-w.7 cxons 3 and 4 are
respectively
ogy search between human, mouse and chicken at the aa level revealed the presence of a highly-conserved segment of 22 aa in the JM domain between mouse and human (100 y,, homology); the chicken sequences differ only in four aa within this segment (Fig. 2). The D. melanogaster sevenless sequences show patchy homology in this segment. In contrast, the entire kinase domain of D. melanogaster exhibits 70% homology to the rat and human c-ROS sequences (Matsushime and Shibuya, 1990; Birchmeir et al., 1990). Identification of the conserved 22-aa segment in the JM domain of c-ros is suggestive of a critical role for it in signal transduction and down regulation.
Birchmcicr, of ROSl
C., O’Neill, K., Riggs, M. and Wigler, M.: Characterization cDNA
from a human
glioblastoma
cell line. Proc.
Acad. Sci. USA 87 (1990) 4799-4803. Chen, J.. Hcllcr, D.. Peon, B., Kang, L. and Wang. oncogenc c-YO.Tcodes for a transmembrane sharing sequences and structural homology Drosophilu melunogusrer. Oncogenc Matsushime,
Natl.
L.H.: The proto-
tyrosine protein kinasc with .srve&s.c protein of
6 (199 1) 257-264.
H., Wang, L. and Shibuya,
M.: Human
c-ROSI
gene ho-
mologous to the v-rus sequence of UR2 sarcoma virus encodes a transmembrane receptorlike molecule. Mol. Cell. Biol. 6 (I 986) 30003004. Matsushimc, H. and Shibuya, M.: Tissue-specific expression of rat c-ro.\-I gene and partral structural similarity of its prcdictcd products with .wv protein of Drosophilcr melunogusrer. J. Virol. 64 (1990) 2 Il72125. Nagarajan.
L., Louie, E., Tsujimoto,
Y.. Baldurzi,
P.C., Huebner,
K. and
Croce, C.M.: The human c-ros gene (ROS) is located at chromosome REFERENCES
region 6q16-6q22. Proc. Natl. Acad. Sci. USA 83 (1986) 656X-6572. Ullrich, .4. and Schlessinger, J.: Signal transduction by rcccptors with
Basler, K. and Hafen, enless protein (1988) 299-311.
E.: Control
requires
of photoreceptor
a functional
tyrosine
cell fate by the sev-
kinase domain.
Cell 54
tyrosine
kinasc
activity.
Cell 61 (1990) 735-738.
