Sequence of a rat cDNA encoding the ERK1-MAP kinase

Gene. 120 (1992) 297-299 0 1992 Elsevier Science Publishers

GENE

B.V. All rights reserved.

297

0378-1119/92/$05.00

06680

Sequence of a rat cDNA encoding the ERKl-MAP

kinase*

(Recombinant DNA; phage il library; signal transduction; tyrosine phosphorylation; microtubule-associated protein 2 kinase; extracellular signal-regulated kinase)

Betina Marquardt

transcription factor phosphorylation;

and Silvia Stabel

Max-Delbrlick-Laboratorium in der Max-Planck-Gesellschaft. D-5000 Cologne 30. Germany Received

by R. Padmanabhan:

18 May 1992; Accepted:

28 May 1992; Received

at publishers:

26 June 1992

SUMMARY

Mitogen-activated protein (MAP) kinases are cytoplasmic and/or nuclear protein kinases which are activated by one or several signal transduction pathways from the cell surface into the nucleus. Their activity is regulated by phosphorylation on Tyr as well as on Ser/Thr residues. A cDNA encoding the rat ERKl member of the MAP kinase family was isolated and sequenced. The longest cDNA consisted of 1875 nucleotides and coded for a polypeptide of 380 amino acids with a predicted M, of 42987.

INTRODUCTION

Protein phosphorylation and dephosphorylation is an essential mechanism for controlling cellular functions in response to extracellular signals. Since the early discovery that oncogenic protein kinases and growth factor receptors share the properties of protein Tyr kinases, the modification of cellular proteins on Tyr residues is thought to be intimately involved in the transduction of mitogenic signals into the nucleus. Two potentially important Tyr kinase substrates of 42 and 44 kDa M,. were originally identified in mouse fibroblasts stimulated with a variety of agents (e.g., Cooper and Hunter, 1985). One of these substrates was subsequently identified as the activated form of MAP ki-

Correspondence to: Dr. S. Stabel, Max-Delbrtlck-Laboratorium, LinnC-Weg

10, D -5000 Koln 30, Germany.

Carl-von

Tel. (49 - 221) 5062 615;

nase, which preferentially phosphorylates the microtubuleassociated protein MAP-2 or myelin basic protein in vitro (Ray and Sturgill, 1987; Rossomando et al., 1989). In its activated form the lcmase is phosphorylated on Tyr and Thr and this phosphorylation is required for enzyme activity (Anderson et al., 1990; Boulton and Cobb, 1991). Considering the apparently wide variety of signals that can activate this kinase Cobb and coworkers have previously re-named MAP kinase as ERK (Boulton et al., 1990). Through biochemical as well as cDNA cloning data it appears now that there exists a family of MAP/ERK kinases (Rossomando et al., 1991; Boulton et al., 1991) the functions of which still have to be elucidated. However, the finding that at least some of the family members are able to phosphorylate and activate the transcription factor c-jun suggests that this family of protein kinases may play an important role in the direct control of transcriptional activity of genes (Pulverer et al., 1991).

Fax (49 - 221) 5062 613. * On request,

the authors

the conclusions

reached

Abbreviations:

aa, amino

signal-regulated nt, nucleotides.

kinase;

will supply detailed experimental

evidence for

in this Short Communication. acid(s);

bp, base pair(s);

MAP kinase,

EXPERIMENTAL ERK,

mitogen-activated

AND DISCUSSION

extracellular-

protein

kinase;

The recent cloning of cDNAs for ERK/MAP kinases revealed at least three distinct members of the MAP kinase

298 “et

1

1 COO Oco =A= =CG=CA

73 LYS

IlP

Ala

Ala

Ala

Ala

Pm

Gco OCGDCOOCTCCG

GLY Gly

Gly

OujooC

uiDQicGGowLGCCCA==GACT

Gly

Gly

01”

Thr

Al.

Gly “al "11 Pro Y.1 Y.1 Pm Oly G1u ",I cl" V.1 ".l OfT %G=TC=IC CCOGTOGTC CCC OO=WLGG~ WL=GTOG~G

plot “11

S-1

Ser

Ala

WA

GCA TAT GAC CAC On;

CGC AA0 ACC AWL G,“J OCT ATC MO

Se= PI0

Cl”

Thr

G1”

Am

“11

Aq!

TAC l-02 CA’S CGC AC0 CTG AWL GAA ATC CAG ATC TTG CTC GGA TYC CGC CAT UG

MT

GTC ATA GGC ATC CGA GAc ATC CTC AGA OCA CCC ACC

ybs

Hi,

Gl”

211 AAG ATC AGC CCC TTC GAG CAT CAA ICC

Tyr

113 I..” 361 CW

01” Al. ML GAA GCC AM

153 Anl 481 Ca;

GlY Is” LYS Ty= Il. Hi* SOI Al. AS” GGC CTC AAG TAC ATA CAC Tee GCC MT

193 ASP Pi-0 Gl”

Gl”

Arg

Thr

L.”

Aq

01”

110 Gl”

A~rg tip “I1 Tyr Ile “.l Gl” Alp Is” Met 01” Thr Asp Lo” ,‘yr AGA GAT OTT TAC AFT GTT CA0 GAC CTC ATG GAG ACG GA‘? CTG NC

Phe ArG His

Tie

Gly

Arq

11s

ly,

Am

Thr Arg

Ile

“al

lo”

Al.

Arg

Al,

Ser Aan As&, Ris Ilo Cys Tyr Phe Ia” Tyr AGC AAT GAC CAC ATC TDC TAC TTC C,“c TX

Lys Is” le” lys G*z Gl” Gin Lo” AA0 CTG CTA AAG AGC CA0 CA0 CM

11~ lys

Pro

Thr

Gin 11s cAG AK

IS” CTC

V.1 LO” “is Ar(l ASP U” 020 CT0 CAC CM: GAC Cllj

lys PI” 201 AS” AA0 CCC KC MT

La” le” 114 AS” Th= Thr Cys ASP ZQ” ly‘ I10 Cys A* P,m Gly Ie” Al, AtG TIP Al. CT-3 CTT AX AAC ACC ACC TGC GAC CTT AAG ATC TOT GAT TTT OOC CT2 GCC Coo ATT GcT

Thr

ArG Trp

Al.

Gly

Tyr

Trp

Sax

V.1

AA0

oa:

TAC ACC AAA TCC A’lT

GAC ATC Too

TCt

GTG

233 Gly

“et

AS”

110 I,.”

Ilo

Lo”

Gly

01”

Am

Cy,

721 GGC RX

Is” CTG TCT CC2 GCT CAT DOT

Ser

Arg

Cl”

Tyr

Pro

“al

Al.

T1.a Pho

ATT CT0 GCT GAG ATG CT.2 TcX ARC COO CCT ATC TX

313 Thr yhs AS” 361 ACC TTT AX 3.53 1081 1201 1321 1111 1561 1681

Le”

,.a”

Gly

“11

CTT AAC KC

01”

Pho

,a”

Ris

CTG ACC GAG TAT GTG G-CC ACA CGC TOG TAC CGA GCC CCA GAG ATC AN

11.

TAT Gly

IS”

Asp

ACT Gac ITT

11~ Lo”

AOP Ais

Il*

Tyr

601 GAC CCT GAG CAC GAC UC Cyr

“is

Cy,

Giy

Gly

CCA CGC TAC ACG CAG CTG CAG TAC ATC GGC GAG Ooc GCG TAC OGE ATC GTC AZ

AT= Tyr Thr Oh

Tyl:

Aq

G4C GTG Dot

PI0 Phs A=P VI1 GlY Pm

Lo" Gl" Tyl: I1a Gly 01" Gly Al.

Pm

CA= CCA T’E

33 LYS =lY =h 121 AAG GiX

Al11 Ala

DCAGT==AGATGEGGCG

ASP MT GCT CGT CT0 TTY UT

A,P GAT CTA I-CA GTA CCC Xc3

Gly

His

Tyr

lrrg

La”

Pro

ASP Gl”

Cl”

Ile

EM, IO”

LO” AS”

CCC GGC AAG CAC TAC CTG GAC CA0 CX

HII

AK

As”

SST ly8

Gly

Thr

Lys

8ar

Ser

Pro

Ser

CAC AT2 CTA OGT ATA CFO OOT TCC CCA XC

11~ As,,

Gin

Ile

A.rp Is”

CAA GAG GAC CTA AAUT TOT

V.1 01” Gl" Ala Is" Ala Ais Pro Tyr le" 01" Gl" Tyr Tyr AS,,Pro Thr Asp Gl” Pro “al Al. Gl” 01” Pro Phe Thr Pho A,* Mot 01” GTA GAG GAA WA CTG GCT CAC CCT PC CTG GAA CAC TIC TAT GAT CCG ACA GAP GAA CCA GTG GCT GAG GAG CCA TTC ACC TTT GAC ATG GAG

le” CTC CCT AGA GAC TGA ATC

lys AAG AIT AGT (100 TCT Too

Pro CCC GCT Ooc GGT CA0 TOO

Fig. 1. The nt sequence

lys AAG CC2 TPC TCT MC AGG

01” GAG TCT TCC GGA ATT Coo

Ar.g CGC CTG CAC AI’G CC2 GGC

,,a” Cl-J CAG TCC GAA AA4 CCG

Cl” GAG GYT CCC CTA CAA A01

%A

GGC COO TAG Coo CCC CM: CGC GCC GGG CCC OOG TCT TCC COG AGT COO OTT DCT TGG

of the ERKl-encoding

by hybridisation

lo” CT0 AGA CT6 TGA Go0 GCC

110 AK AAA CCC CCG CTA COT

Phe TX TGA CAT CCC OTT GCC

cDNA

with a fragment

Gin CM ACT CTC ,‘A0 Tee CCT

01” GAG TTC CCC GAC CT0 CCC

2.0 (USB, Cleveland,

OH). The sequence

Thhr Ala ACA GCC CTC AAC 2% ACC CTG TGC AGC AGC UC CTC

and deduced

corresponding

Five different clones were plaque puritied and the pBluescript Sequenase

ly,

Tyr

Pm Am lya ArG 11s Thr CCA AAC AAG CGC ATC AU

1801 AGC CCG TX% ACG Or0

CA) was probed

Pro

Thr

encoded

Arg COC COO WA TAC CAG CCT

Pho TTC GAC GTO AGA CC2 GTG

aa sequence.

to nt 22-691 plasmid

Gin CA0 CCC ATC MO AGG CC2

Pro CCA GGC AGG GTG CC2 GTA

Al. GCA KA ICC ‘SC ‘XT AAT

Methods.

Pro CCA GGC CAG ACT CTC ATA

01” GAG TGG A= GAG CC2 TAA

Ala OCC ACC lIZA TAG CTC ATA

Pm CCC AAG TCT GCT AA0 TAG

TEA TM GGT CTG AA0 CTG AWL

CM GAA Go0 CCT CT-G CTG CTC EC CCA CAT TGT GTA

CAG Ooc TOT CCT GTA TAA

AWL ACC CTT ACT ACG AAA

CCC CCT TAT CAT Ccc AAG

CT0 CTC CTA CCT TTG XC

TCC ACT TCC OTT CTG TTC

TTT CrO CT-S GGA CT2 TGA

WG CT0 CTA ACC CPG KG

ACC GGG GCC CCA TOT AGG

3*0 TGG KT CCA CCC CTG CCC

11175

A rat brain cDNA

of the published

was rescued.

a polypeptide

Gly GGG AGC TOO GAG AAC TCT

partial cDNA

library in ZAP11 sequence

(Stratagene,

for ERKl

(Boulton

The longest clone of 1875 nt was sequenced

of 380 aa with a predicted

M, of 42987.

La Jolla,

et al., 1990).

on both strands

EMBL databank

using

accession

No.

X65198.

family in vertebrates, ERKl, 2 and 3, based on sequence comparison (Boulton et al., 1991). For the MAP kinase ERK2, full-length cDNA clones have been isolated from several species: rat (Boulton et al., 1991), Xenopus (Gotoh et al., 1991), mouse (Her et al., 1991) and human (Owaki et al., 1992). However, only partial sequence information is available for the MAP kinase member ERKl (Boulton et al., 1991; Crews et al., 1991; Owaki et al., 1992). In order to analyse the structure/function relationship of this enzyme we set out to isolate a full-length cDNA coding for the ERKl member. The cDNA described here was 1875 nt long and encoded a polypeptide of 380 aa with a calculated M, of 42 987 (Fig. 1). Interestingly, the ERKl coding sequence starts with a stretch of six Ala which are also found in the ERK2 members. The significance of this stretch is unknown; a search in the SwissProt database identified only two other proteins with the same six Ala start. The rat ERKl sequence is 97% homologous to the published partial ERKl sequence from man (Owaki et al., 1992) and shows 87% identity to the p42 ERK2 kinases from different species (Fig. 2).

REFERENCES Anderson,

N.G.,

Ma&r,

ment for integration pathways

This work was supported by a grant from the Ministry for Research and Technology (BMFT).

of signals

for activation

N.K. and Sturgill, T.W.: Requirefrom two distinct

of MAP

kinase.

Nature

phosphorylation 343 (1990) 651-

653 Boulton,

T.G.

and Cobb,

signal-regulated Regulation Boulton,

M.H.:

kinases

T.G.,

Nye, S.H.,

Morgenbesser,

SD.,

and Yancopoulos,

extracellular

antibodies.

Cell

T.G.,

D.J., Ip, N.Y., Radziejewska, R.A., Panayotatos,

E.,

N., Cobb, M.H.

G.D.: ERKs: A family of protein-serine/threonine

insulin and NGF. Moomaw,

of multiple

with antipeptide

Robbins,

DePinho,

kinases that are activated Boulton,

Identification

(ERKs)

2 (1991) 357-371

and tyrosine

phosphorylated

in response

to

Cell 65 (1991) 663-675

Yancopoulos,

G.D.,

Gregory,

J.S.,

Slaughter,

C., Hsu, J. and Cobb, M.H.: An insulin-stimulated

kinase similar to yeast kinases involved in cell cycle control.

C.,

protein Science

249 (1990) 64-67 Cooper, J.A. and Hunter, protein tyrosine

T.: Major substrate

for growth factor-activated

kinases is a low-abundance

protein.

Mol. Ceil. Biol.

5 (1985) 3304-3309 Crews, C.M., Alessandrini, A.A. and Erikson, R.L.: Mouse ERK-1 gene product is a serine/threonine protein kinase that has the potential to phosphoprylate

on tyrosine.

Proc. Natl. Acad.

Sci. USA 88 (1991)

8845-8849 Devereux,

F., Haeberli,

sequence analysis (1984) 387-395 ACKNOWLEDGEMENTS

J.L., Tonks,

P. and Smithies, programs

0.:

A comprehensive

for the VAX. Nucleic

Acids

set of Res.

Gotoh, Y., Moriyama, K., Matsuda, S., Okumura, E., Kishimoto, Kawasaki, H., Suzuki, K., Yahara, I., Sakai, H. and Nishida, Xenopus M phase MAP kinase: isolation of its cDNA by MPF. EMBO J. 10 (1991) 2661-2668

12 T., E.:

and activation

Her, J.-H., Wu, J., Rall, T.B., Sturgill, T.W. and Weber, M.J.: Sequence

299

rE1 hF,l rhE2 hFZ? MPKl

1 mAAAAAPGG

GGGEPRGTAG ..R.E. ... .. ..--- --.------. .... ..-.A ------.-.. . ...G..SNP ...-------

VVPVVPGEVE .G.G...... --------P. --.-----.. --------P.

WKGQPFDVG M......... M.R..V.... M.R..V.... M.R..A....

EGAYGMVSSA YDHVRKTRVA IKKISPFEHQ TYCQRTLREI .......................................... ..... ..C.. ..NLN.V ....................... ..... ..C.. ..NLN.V....................... ..... ..C .. H.N.N.V ....................... VIGIRDILRA .......... I...N..I .. I...N..I .. I...N..I ..

50 PRYTQLQYIG .. ..... .. . . ...N.S... ....N.S... . ..IN.A... 100 QILLGFRHEN ..R ..... K...R ..... K...R

....

K...R.K ...

150 PTIEAMRDVY IVQDLMETDL YKLLKSQQLS NDHICYFLYQ s ....................................... ..I.Q.K ................ ..T.H ............ ..I.Q.K ................ ..T.H ............ ..I.Q.K ................ ..T.H ............

200 ILRGLKYIHS ANVLHRDLKP SNLLINTTCD LKICDFGLAR IADPESDHTG .................................................. ...................... ..I# ............... V...D ..... ...................... ..L ............... V...D ..... ...................... ..L ............... V...D ..... 250 FLTEYVATRW YRAPEIMLNS KGYTKSIDIW SVGCILAEML SNRPIFPGKH .................................................. .................................................. .................................................. .................................................. 300 YLDQLNHILG ILGSPSQEDL NCIINMKARN YLQSLPSKTK VAWAKLFPKS .................................................. ....................... ..L.... ..L...H.N. .P.NR...NA ....................... ..L.... ..L...H.N. .P.NR...NA ....................... ..L.... ..L...H.N. .P.NR...NA 350 DSKALDLLDR MLTFNPNKRI TVEEALAHPY LEQYYDPTDE PVAEEPFTFD ................................................. . ......... . .... ..H... E..Q ........... ..S .. .I..A..K .. .......... .... ..H... E..Q ........... ..S .. .I..A..K .. .P ..... ..K ......H... E..A ........... ..S .... ..A..K .E 380 MELDDLPKER IXELIFQETA RFQPGAPEAP ....................... ..VL... ......... . .... ..E...... ..YRS .......... .... ..E...... ..YRS .......... .... ..E........ .

Fig. 2. The aa sequence comparison of rat ERKl (rE1) to the partial human ERKl sequence (hE1; Owaki et al., 1992), and the ERK2 proteins from rat (rE2; Boulton et al., 1991), human (hE2; Owaki et at., 1992), Xenopus (MPKI; Gotoh et al., 1991) and mouse (mE2; Her et al., 1991). Dots represent aa identities and dashes represent gaps for improved alignment. Alignment was assisted by the programs developed by Devereux et al. (1984).

of pp42/MAP kinase, a serine/threonine kinase regulated by tyrosine phosphorylation. Nucleic Acids Res. 19 (1991) 3743 Owaki, H., Makar, R, Boulton, T.G., Cobb, M.H. and Geppert, T.D.: Extracellular signal-regulated kinases in T cells: characterization of human ERKl and ERK2 cDNAs. Biochem. Biophys. Res. Commun. 182 (1992) 1416-1422 Pulverer, B.J., Kyriakis, J.M., Avruch, J., Nikolakaki, E. and Woodgett, J.R.: Phosphorylation of c-jun mediated by MAP kinases. Nature 353 (1991) 670-674 Ray, L.B. and Sturgill, T.W.: Rapid stimulation by insulin of a serine/ threonine kinase in 3T3-Ll adipocytes that phosphorylates microtubule-associated protein-2 in vitro. Proc. Natl. Acad. Sci. USA 84 (1987) 1502-1506 Rossomando, A.J., Payne, D.M., Weber, M.J. and Sturgill, T.W.: Evidence that pp42, a major tyrosine kinase target protein, is a mitogenactivated serine/threonine protein kinase. Proc. Natl. Acad. Sci. USA 86 (1989) 6940-6943 Rossomando, A.J., Sanghera, J.S., Marsden, L.A., Weber, M.J., Pelech, S.L. and Sturgill, T.W.: Biochemical characterization of a family of serine/threonine protein kianses regulated by tyrosine and serine/ threonine phosphorylations. J. Biol. Chem. 266 (1991) 20270-20275