Expression, Characterization, and Genomic Structure of Carp JAK1 Kinase Gene

(1)

Volume 15,Number 10,1996

Mary

AnnLiebert,Inc.

Pp.

827-844

Expression,

Characterization,

and

Genomic Structure of

_Carp

JAK1 Kinase Gene

MAU-SUN

_CHANG,1

GEEN-DONG

_CHANG,2

JIANN-HORNG

_LEU,3

FORE-LIEN

HUANG,1-3

CHEN-KUNG

_CHOU,4

CHANG-JEN

_HUANG,3

and

TUNG-BIN

LO2-3

ABSTRACT

A

_{3.7-kb cDNA encodes the carp}

_JAK1

kinase of

1,156

amino

acid residues. The overall amino acid

sequence

identity

between carp

JAK1

and

murine

JAK1, JAK2, JAK3,

and

human

TYK2

is

57%, 35.5%, 31.3%,

and

42.4%,

_{respectively.}

In

_addition,

_carp

_JAK1

shows

_higher

_sequence

_homology

to

mammalian

JAK1

in

both

the

kinase-like

_(JH2)

and kinase

(JH1)

domains

_{(approximately}

70%

_identity).

_Therefore,

_carp

JAK1

is

a

ho-molog

of mammalian

JAK1.

To

_investigate

the

_possible

function of

JH2

domain,

_full-length,

and various

trun-cated

forms of

carp

JAK1

were

_produced

in the baculovirus

_system.

Our

results demonstrate

that

c-JHl

and

C-JH2

associate with each other and

C-JH2

can be

tyrosine-phosphorylated

by

c-JAKl

and

by

c-JH(l

+

2).

The JAK

I gene was

also isolated from

a

_carp

_{genomic library}

and characterized. This

_gene

is divided into 24

exons

spanning

atleast

31

kb

of

genomic

DNA. Exon 1 contains

the

5'-untranslated

region

and

exon 2

con-tains the

_putative

translation initiation site. The

2.5-kb DNA

_region

_upstream

of the

_{transcription}

initiation

site contains

numerous

potential binding

sites for

transcription

factors

including

NF-IL6, HNF-5, API,

GHF-5,

and

E2A. When this

DNA

_fragment

was

placed

upstream

of the

chloramphenicol acetyltransferase

(CAT)

reporter

gene

and

transfected into

a carp

CF

cell

line,

it could drive the

_synthesis

of CAT enzyme 16 times

more

efficiently

than

the

promoterless

pCAT-Basic.

Deletion

analysis

defined

a

positive

regulatory

region

be-tween

—1,023

and

—528.

A smaller

_region

_(—181

to

₊₅₉₎

without

any

_typical

TATA-box sequences, G

+

C-rich sequences,

or other

binding

sequences

for

known

_{transcription}

factors still

had

promoter

activity.

Constructs without this

_region

did

not have detectable

_promoter

_activity.

This

_suggests

that

this

_region

of

DNA may

play

an

important

role

in

the

expression

of

carp

JAK

I gene.

INTRODUCTION

of the external

_signals

tothe cell.

_{Upon binding}

of the

_ligand,

thekinase

_activity

of the

_receptor

PTK is

induced,

_resulting

in

TYROSiNE

PHOSPHORYLATION

_{catalyzed by protein tyrosine}

ki-

_{phosphorylation}

of the

_receptor

and cellular substrateson

tyro-nases

(PTKs)

isa

key

_step

in

transducing signals

fromex- sine residues

(Ullrich

and

_{Schlessinger,}

1990).

By

contrast, the ternal stimulitothe nucleus.

Therefore,

PTKs

_play

very

impor-

nonreceptor PTKs,

duetothe lack of extracellular

domains,

can-tantroles in the

_regulation

of cell

_{proliferation}

and differentiation not

_directly

interact with extracellular

ligands.

Therefore,

the

(Hunter

and

_Cooper,

1985;

Hanks et

al,

1988).

On the basis of

_{physiological}

functions of these

_nonreceptor

PTKs remained ob-their structural

similarities,

PTKscanbe divided intotwo

_major

scurefor

_quite

some time until the observation that

_p56lck

was groups:receptorandnonreceptorPTKs.

Receptor

PTKs contain associated with the CD4 and CD8

_co-receptors

(Veillette

et

al,

extracellular, transmembrane,

and

_cytoplasmic

domains. Theex-

1988).

This

_suggests

that

by

binding

nonreceptor PTKs,

some tracellular domainsareabletoassociate with

_ligands,

where the transmembranereceptors

lacking

TKdomainscanactina

man-cytoplasmic catalytic

domains are

responsible

for transmission ner

_analogous

tothereceptorPTKs.

'Department

of

_Zoology,

and2GraduateInstitute of Biochemical_Sciences,National Taiwan

_University,

_Taipei,

Taiwan,

instituteof

_{Biological Chemistry,}

AcademiaSinica,

Taipei,

Taiwan.

4Department

of MedicalResearch,VeteransGeneral

_{Hospital, Taipei,}

Taiwan.

(2)

The

_polymerase

chainreaction

(PCR) (Mullís

and

Faloona,

1987)

has been

_employed

toclone

_potential

PTKs,

using

de-generate

oligonucleotide primers

corresponding

to conserved

peptide fragments

inkinase domains. The JAK

(Janus kinase)

family

wasfirst identified

by

this_strategy

(Wilks,

_1989;

Wilks et

al, 1991).

This

_{family belongs}

tothe

_nonreceptor

PTKs and

currently

consists of JAK1

_(Wilks

et

al, 1991;

_homology

domains SH2 and SH3

(Pawson

and

_{Schlessinger,}

1993)

butbear an unusual feature of

_having

akinase-like domain. The functional role of TYK2 was first demonstrated

_by

its

_{participation}

ininterferon

(IFN)

signal

transduction

_pathway

and its association with the IFN-a

receptor

(Velazquez

et

al,

1992).

_Recently,

_many

cytokine

re-ceptors

have been foundtoassociate with one ortwo or more members of the JAK

_{family, including}

the

_receptors

_for

ery-thropoietin

(EPO) (Witthuhn

et

al,

1993),

_growth

hormone

(Argetsinger

et

al,

1993),

prolactin (Campbell

et

al, 1994;

Da Silva et

al, 1994;

Rui et

al,

1994),

interleukin 2

(IL-2)

M,

leukemia

_inhibitory

factor

(LIF)

(Stahl

et

al,

1994),

granulocyte-macrophage colony-stimulating

factor

(GM-CSF)

(Quelle

et

al,

1994),

granulocyte

colony-stimulating

factor

(G-CSF) (Nicholson

et

al,

1994),

and

IFN-y (Muller

et

al, 1993;

Watling

et

al,

1993).

Although lacking

protein

kinase

domains,

the above members of the

_cytokine

_receptor

_superfamily

can

couple ligand binding

with the

_{tyrosine phosphorylation}

of downstream

effectors,

which is mediated

by

association with the JAKs. The activated JAKs then

_{phosphorylate cytoplasmic}

transcription

factors STATsor the

_signal

transducer and

acti-vators of

_{transcription.}

After

_tyrosine

_{phosphorylation,}

these

transcription

factorsarehomo-or

heterodimerized,

translocated into the

nucleus,

and induce

_{transcriptional}

_responses

(Shuai

et

al, 1993;

Darnellet

al, 1994;

Ihleet

al, 1994;

Schindler and

Darnell,

1995).

In

_fish,

_growth

hormone and

_prolactin

from many

species

have been isolated and cloned

_(Chang

et

al, 1992a,b;

Bemardi et

al,

1993),

whereas

only

oneIFN_{from flatfish has been}

pu-rified and cloned

(Tamai

et

al,

1993).

Basedon a

_variety

of studies from

mammals,

the JAK

_family

membersareshownto

be involved in the

signal

transduction of

_growth

hormone

(Argetsinger

et

al,

1993),

prolactin (Campbell

et

al, 1994;

DaSilvaet

al,

1994),

and interferons

(Velazquez

et

al,

1992).

Therefore,

as aninitial

_step

tounderstand the

_signal

transduc-tion in

fish,

westartedto_{isolate the carp JAK kinase}

_family

_by

PCR. As

_reported

in this paper,amember of the JAK

_family

wascloned and

_designated

_{carp JAK1 kinase. The carp JAK1} kinase has a

_higher

_sequence

_homology

in both JH1 and JH2 domains

(70%

identity)

tohuman and murine JAK1

(Wilks

et

al, 1991;

_Yang

et

al,

1993).

Because the function of the JH2 domain is

unknown,

we

explored

its

_activity

inabaculovirus

expression

system,which has been usedto_{express mammalian}

JAK2 kinase

(Quelle

et

_al,

1994;

Duhe and

Farrar,

1995).

We made constructs that encode

_full-length

JAK1

(c-JAKl),

the JH1 and JH2 domains alone

(c-JHl

and

c-JH2),

and the JH1 and JH2 domains in combination

[c-JH(l

+

_2)]

and

_expressed

them in the baculovirus system. Coinfection and

immunopre-cipitation experiments

showed thatc-JHl and C-JH2 could as-sociate with each

other,

and that C-JH2 could be

tyrosine-phos-phorylated by

c-JAKlor

c-JH(l

+

2).

Our results_may

_provide

clues

_concerning

thefunction of the JH2 domain.

Studies

_by

others showed that

_expression

ofmostmembers of the mammalian JAK kinase

_family

suchas

_{JAK1, JAK2,}

and

TYK2,

is

_ubiquitous

whereas

_only

JAK3 is

_{mainly expressed}

in

_{hematopoietic}

cells

_(Ihle

and

Kerr,

1995).

As afirst

_step

to

explore

the molecular basis

_underlying

the

_regulation

_{of carp} JAKl gene

expression,

we also cloned and characterized the carp JAKl gene.We have

mapped

the carp JAKl

transcription

start site and characterized its

_promoter.

With nested deletion

mutantsof the

_5'-flanking

_region

fusedtothe

_{chloramphenicol}

acetyltransferase

(CAT)

reporter gene, the promoter

_activity

was characterized

by

transfection into afish cell line. To our

knowledge,

this is the first

_report

tocharacterize the

_genomic

structure and the

_promoter

region

of JAK kinases.

MATERIALS AND METHODS

Materials

Insect

Spodoptera

frugiperda

(Sf9)

cells were _{grown in} Grace's medium

_containing

10% fetal calf serum

(FCS;

Life

Technologies,

Inc.,

Gaithersburg,

MD).

Monoclonal

anti-phos-photyrosine IgG

clone 4G10 was

_purchased

from

_Upstate

Biotechnology

Inc.

_(Lake

Placid,

NY).

Construction

of

a

random-primed

cDNA

_library

Total RNAwasisolated from carp brain

by

the

guanidinium

isothiocyanate

method

_(Chomczynski

and

Sacchi,

1987),

fol-lowed

_by

the use of a

QuickTrack

mRNA isolation kit

(Stratagene,

La

Jolla,

CA).

The isolated mRNA was used for the

_synthesis

of

_{random-primed}

cDNA.

Amplification of

DNA

_fragments

_by

PCR

Degenerate

primers

(Walks, 1989)

were

_designed

tofit the amino acid sequences thatare conserved among PTKs

(Hanks

et

al,

1988; Hanks,

1991).

The amino acid sequences of the

two

_{opposing primers}

are HRDLAAR

(subdomain

VI in

Fig.

2,

below)

and DVWSFG

(subdomain IX).

The first-strand carp brain cDNA was usedas

template.

The conditions for the re-action were94°C for 1

min,

45°C for 1

min,

and 72°C for 2 min in 30

_cycles.

cDNA

_{library screening}

An

_{oligo-(dT)-primed}

_{carp liver cDNA}

library

in the lamda ZAP II

(Short

et

al,

1988)

obtained from

_Stratagene

was screened

_{by using}

a

_{PCR-amplified}

DNA

_fragment

as a

_probe

(see Results).

The

_probe

was labeled

_using

a DIG DNA

Labeling

Kit

_(Boehringer

Mannheim, Mannheim,

Germany).

Hybridization

and

_washing

werecarried out

_according

tothe standard

_procedures

(Sambrook

et

al,

1989).

Signals

were de-tected

_using

the DIG Luminescent Detection Kit for Nucleic Acids

(Boehringer Mannheim).

Putative

_{positive plaques}

were

purified

through

several rounds of

_rescreening

atlower densi-ties and then excisedas

pBluescript plasmids according

tothe manufacturer's instructions. To isolate the

_full-length

cDNA,

(3)

FISH TYROSINE KINASE

JAKl

the

_300-bp

DNA

_fragment

from the5' end of

_{pJ21 (Fig.}

1)

was

amplified

by

PCR, DIG-labeled,

and then usedas a

probe

to

screenanother

_{random-primed}

_{carp brain cDNA}

library

as de-scribed above.

Sequence

analysis

cDNA clonesweresubclonedinto

plasmid pUC18

(Yanisch-Perron et

al,

1985)

and

sequenced by

the

dideoxy

chain-terminationmethod

_(Sanger

et

al,

1977)

using

a

_Sequenase

kit

(United

States Biochemical

_Corp.

Cleveland,

OH).

Several pro-grams from IntelliGenetics

(Mountain

View,

CA)

wereusedto

analyze

the nucleotide sequences.

DNA

constructions

DNA

_{fragments encoding}

the entire carp JAKl were

ampli-fied with

_overlap

extension PCR

(Ho

et

al,

1989)

fromJ9 and J21 cDNA

_(Fig.

1)

by using

the

_{following primers: primer}

F1.5'-ATATCAGGATCCATGCCAGAACTAGCAGTCATG-3' and

_primer

Rl,

5'-GTGGAAACTCAGCTGGCTGAC-3';

primer

F2,

5'-GTCAGCCAGCTGAGTTTCCAC-3'and

_primer

R2,

5'

-CAGTAC

AAGÇTTCTC

ATCTTATTTCCAT

AGTTA-3'. _{The nucleotide sequences} of

_primers

Rl and F2 are

com-plementary

and

they

are locatedatthe end of clone J9. Other

DNA

_{fragments encoding}

the kinase-like

domain,

kinase

do-main,

and both domainswere

amplified

with the PCR from J21 cDNA

by

using

the

_{following primers:}

c-JH2

(residues

572-875),

5'

-ATATCAGGATCCCAGCTGAGTTTCC

ACC-GCATC3'(F3)

and

5'-CAGTACAAGCTTGAGGAACCTC-TTCTCAAACAC-3'(R3);

c-JHl

(residues 869-1,156),

5'-ATATC

AGGA1ÇCGTGTTTG

AGAAGAGGTTCCTC-3'

(F4)

and 5'-CAGTAC

AAGÇTTCTC

ATCTTATTTCCAT

AGTTA-3'(R2); c-JH(l

+

_{2) (residues 572-1,156),}

5'-ATATCA-GGATCCCAGCTGAGTTTCCACCGCATC-3'

(F3) and

5'-CAGTACAAGCTTCTCATCTTATTTCCATAGTTA-3'(R2).

Each

_primer

encodesa

unique

restrictionsite

(underline) (Bam

HI, GGATCC;

and Hind

III,

AAGCTT).

The PCR

_products

bases o carp JAKl

s'-f

—n 3500 3730

K

J9 J21 HI

FIG.1. Restriction maps of carp JAKl cDNA clones. The ki-nase-like

(JH2)

and kinase

(JH1)

domains are

_represented

by

the

_{horizontally striped}

and black

boxes,

respectively.

Restriction enzyme sites are _{shown above carp JAKl cDNA.} The

_poly

(A)

tail is

_{represented by}

AAA.The cDNA

clones,

Jl 1 and

J21,

were isolated from a_{carp liver}

_library

whereas the clone J9 wasisolated froma_{carp brain}

library.

werethen restricted with Bam HI and HindIIIand

ligated

into

pQE30

(QIAGEN

Inc., Chatsworth,

CA).

DNA_sequence

analy-sis was

performed

to _{confirm the accuracy of the PCR}

prod-ucts.

Expression

in

Escherichia coli

All the

_{resulting plasmids (pQEJAKl, pQEJH(l}

+

_2),

pQEJHl,

and

_pQEJH2)

were transformed into E. coli strain JM109. Thetransformantswere_grown

overnight

at37°C in LB broth and diluted 1:10 into fresh medium. After incubation at

37°C for1

hr,

isopropyl-/3-D-thiogalactopyranoside

(IPTG)

was added to a final concentration of 1

mM,

and incubation was continued for 3 hr. Cellswerecollected

_{by centrifugation.}

Due

toall the recombinant

_proteins

containastretchof six

histidines,

therefore these

_proteins

canbe

purified by using

Ni2+-nitrilo-triacetate-agarose (Ni-agarose) (QIAGEN

Inc.)

according

tothe

procedures

asdescribed

(Huang

et

al,

1995).

Antibodies

Purifiedrecombinant

_protein

c-JHl orc-JH2wasdissolved in

_phosphate

buffersaline

(PBS)

and mixed

_thoroughly

withan

equal

volume of Freund's

_{complete adjuvant}

for the first

in-jection

of Freund's

incomplete

adjuvant

for the second and third

injection. Approximately

100 pg of recombinant

protein,

for each

_injection,

was

subcutaneously injected

into the back of

guinea pig.

Expression

in

the

baculovirus

_system

All of the

_pQE30

constructs described in the E. coli

ex-pression

systemwere

_digested

with EcoRIand XbaIand then

ligated

into the transfer vector PVL1393

_(PharMingen,

San

Diego,

CA).

Plasmids

(0.5

pg of

each)

were co-transfected into Sf9 cells with 0.1 pg of

"Baculogold"

virus DNA

(PharMingen),

whichcontainedalethaldeletion.The

parental

baculovirus is unabletoinfect insect cells and

_only

recombi-nant baculovirus

_containing

the

_plasmid

constructcan

repli-catein Sf9 cells.

Sf9 cells were infected with various baculovirusat a

multi-plicity

of infection

(moi)

of 10

_{plaque-forming}

units/cell.

After 60

hr,

cells were

harvested, washed,

and

lysed

inabuffer

con-taining

150 mM

NaCl,

1% Triton

X-100,

and50 mM Tris

_pH

7.5. The Triton-insoluble cell

_pellets

weresolubilized inabuffer

containing

1.5%

_{/V-lauroylsarcosine,}

25 mM

triethanolamine,

and 1 mM EDTA

_pH

8.0

(Frankel

et

al,

1991).

Both Triton-soluble and -insoluble fractions were incubated with the

Ni-agarose.

After extensive

_washing

with 0.5 M

NaCl,

proteins

wereeluted

by

0.1 M EDTA

_pH

8.0. An

_equivalent

amountof

_protein

was

subjected

to

NaDodS04-polyacrylamide

gel electrophoresis

(PAGE),

blottedto_{nitrocellulose paper, and}

_probed

with differ-entantibodies.

Western

blot

The methodofTricine

NaDodSGj-PAGE (Schagger

andvon

Jagow, 1987)

wasused. The

_gel

concentrationwas7.5%. After

NaDodS04-PAGE,

the

_proteins

were

_analyzed

by

immunoblot

using

anti-PYmAbor

polyclonal

antibodies

specific

for JH1 or JH2 domain

according

tothe

_procedures

asdescribed

(Huang

et

(4)

Immunoprecipitation

Sf9 cellswerecoinfected with vAcJH2 and vAcJHl. After 60

hr,

cellswerecollectedand solubilized

by

resuspension

and incubation in the

_{immunoprecipitation}

buffer

(50

mM Tris

pH

7.4,

150 mM

NaCl,

1 mM

_Na3V04

and 1%

NP-40).

Immunoprecipitation

was

performed

by

the addition ofanti-JHl oranti-JH2 antiserumatadilution of 1:100orwith

_preimmune

serum as a control reaction. The mixture

(200

pi)

was incu-batedat4°C for 2 hrfollowed

_by

anincubation with 50

pi

of 50

_{mg/ml slurry}

of Protein

_A-Sepharose

(Pharmacia,

Uppsala,

Sweden)

for 1 hr. The

_Sepharose

was collected and washed three times with the

_{immunoprecipitation}

buffer andtwotimes with 20 mM Tris

_pH

7.4,

1 mM

_EDTA/1

mMEDTA.

_Samples

werethen boiled ina0.5%

NaDodS04

_sample

bufferto

_disrupt

the

_complexes.

Productswere

subjected

to

NaDodS04-PAGE

and

_{analyzed by}

immunoblot

_using

anti-PY mAbor

polyclonal

antibodies

_specific

forthe JH1 orJH2 domain.

Reverse

_{transcription}

and

PCR

mRNA

(0.5 pg)

isolated from carp brain and liver tissues wasincubatedat65°C for 5 min inabuffer

containing

50 mM

Tris-HCl,

75 mM

KC1,

3 mM

_MgCl2,

10 mM

dithiothreitol,

2 units of RNasin and 1.25 mM

deoxynucleotide

triphosphatates

(dGTP,

dATP, dTTP,

and

dCTP),

and then

_kept

onice. Two hundred units of

_{Superscript Moloney}

murine leukemia virus reverse

_{transcriptase}

_(RT;

GIBCO

BRL,

Gaithersburg,

MD)

and

oligo(dT)i2_i8 primer

(2 pg)

and random hexamer

_primers

(2

pg)

wereadded and incubatedat 37°C for 1 hr. Thereaction was then

stopped by

incubationat95°C for 5 min.

Fivesets of

_{specific primers,}

Fl/Rl, F1/R2, F3/R3, F4/R2,

and

F3/R2

were_{used and their sequences}weredescribed in the DNA Construction section. PCRwasthen carriedout

_by

addi-tion of the heat-treated RT

mixture,

PCR

buffer,

and

_Taq

poly-merase

(Promega,

Madison, WI).

Only

the

amplified

DNA

frag-ments

_by

_F3/R3

and

F4/R2

from brain tissues and PCR

_product

by

Fl/Rl

from liver tissueswereisolated froman_agarose

gel

and subcloned into the

_pGEM-T

vector

(Promega).

Other

ana-lytical

methods were the same as described in the section of

Sequence Analysis.

Isolation

_of

_carp

JAKl

_genomic

clones

A

commercially

available carp liver lambdaFIX II

genomic

library (Stratagene)

was usedto isolate 15-to 18-kb

genomic

DNAclones

_containing

the gene that encodes carp JAKl.

Full-length

carp JAKl cDNA was labeled

_using

a DIG DNA

Labeling

Kit

_(Boehringer

_Mannheim).

_{Approximately}

1 X

106 independent

cloneswere

plated

ata

density

of 5 X

104 _plaque

forming

units/150-mm

Petri dish.

_{Hybridization}

and

_washing

werecarriedoutasdescribed above.

Signals

weredetected

us-ing

the DIG Luminescent Detection Kit for Nucleic Acids

(Boehringer

Mannheim).

Putative

_{positive plaques}

werefurther

purified through

several rounds of

_rescreening

atlower densi-ties. Four distinct carp

genomic

DNAclones

_(designated

Jl, J2,

J3,

and

J4)

were

isolated,

and DNAwas

_prepared

from each clone. These carp

genomic

clones were

digested

with various restriction endonucleases. The DNA

_fragments

were fraction-ated

_{by electrophoresis}

in 1% agarose

gels

and transferredto

nylon

filters

_(Sartorius

AG,

_{Goettingen, Germany).}

The

result-ing

DNAblotswere

_probed

under theabove conditions of

_high

stringency

with the DIG-labeled whole carp JAKl cDNA.

Sequence analysis

of

genomic

clones that encode

carp JAKl

Phage

DNA was

digested

with Not

I,

Sac

I, 5a/1,

and Xba

I and subcloned into either the

_pUC18

or

pBluescript

vector.

_primers

that are from two

differentexons.The

_genomic

DNA_sequencewas

analyzed

with the Genetics

_{Computer Group}

_{software program. The}

tran-scription

factor

_recognition

site data bases

(releases

7.3 and

6.5)

wereusedto

_{identify potential transcription}

factor motifs within the

_{5'-flanking region}

_{of the carp JAKl gene.}

Primer extension

_analysis

An antisense

_{oligonucleotide}

(5'-GlTT

AATTTCGATCAT-TACAGACGTTTG-3')

complementary

to the 5' _{end of carp} JAKl cDNA clone J9

_(Fig.

1)

was labeledatthe 5'end with

["y-32P]ATP

by

T4

_{polynucleotide}

kinase and

_purified

on a

Stratagene

NucTrap probe purification

column. The labeled

primer

was annealedto5_pg of

_poly(A)+RNA

_prepared

from adult carp liver totalRNAand extendes with

Moloney

murine leukemia virus

(M-MLV)

reverse

_{transcriptase}

(GIBCO BRL)

asdescribed

_previously

(Sambrook

et

al,

1989).

The extended

products

were

analyzed

on a5%

polyacrylamide,

7Murea

se-quencing

gel.

The size of the extended

products

was inferred froma

_sequencing

ladder of the JAKl gene obtained from the same

primer

used for

primer

extension.

Plasmid construction

_for

CAT

_functional

_analysis

A 3-kbSal I

_{fragment encompassing}

for

_5'-flanking

_region

was subcloned into

_pBluescript

and various deletion mutants

were

generated

from this clone

by

PCR

using specific primer

FIG. 2. _{Amino acid sequence}

_alignment

of the carp,mouse

(Yang

et

al,

1993)

and human

(Wilks

et

al,

1991)

JAKl

_proteins.

Alignments

were

_initially

made

_by

computer

analysis

andwere

_{subsequently aligned by inspection. Gaps}

areintroducedto

op-timize

_alignment

and shownas dots. The identical residuesare

represented by

dashes. The

putative

kinase domains are delin-eated witharrows. Subdomains Ia-XIa of the kinase-line domain and subdomains I-XI of the kinase domain aredenoted

ac-cording

to the

_previous

_reports

(Hanks

et

al, 1988; Hanks,

1991).

The sequence of carp JAKl has been

deposited

in the

(5)

JAKl

cJAKl MPELAVMELGRQLCGKMKKQRKAEMTVLTVMKGLEIHFYLPDTHQLEYFKDCHTAEDL 58 mJAKl MQYLNIKEDCNAMAF-A--RSFK-T-VKQWPEP-V-VT-L-REP-RLGSGEY-E- 60 hJAKl MQYLNIKEDCNAMAF-A--RSSK-T-VNLEAPEP-V-VI-S-REP-RLGSGEY-E- 6 0 cJAKl CVEGCQEDATSHLCADNLFALSEESQDLWYAPNHAFKITEETSIKLHYRMRFYFTNWHAP 118 mJAKl -IRAA-ECSI-P--H.-YD--TK-RIITVDDK--LR-GT 119 hJAKl -IRAA-ACRI -P- -H.-YD-NTK-RTITVDDKM-LR-GT 119 cJAKl VRTESPVWRHSLFKHKGVSVSPKGPEGTPLLDAASLEYLFAQGQYDFLRGLAPVRAPQNE 178 mJAKl NDN-QS-PK-Q-NGYEKKRV-EA-S-LIKC-I-D-KT- 17 9 hJAKl NDN-QS-PK-Q-NGYEKKKI-DA-S-LVKC-I-D-KT- 179 cJAKl AEKHEIENECLGMAVLAITHHAKSNDLPLSGVGAETSYKRFIPDSLNRTIKQRNFSHVYV 23 8 mJAKl QDG-D-S-MMKKMQ-PELPKDI-Y--ET--KS-R-LLTRMR 23 9

hJAKl QDG-D-S-MMKKMQ-PELPKDI-Y--ET- -KS-R-LLTRMR 23 9

cJAKl YNNVFKNFLNEFNSKTIQDSNITLYDLKVKYLSTLETLTQGLGRETIEPKILKVSGESDG 2 98

mJAKl I-D--K-N-C--SVSTH-A-KHY-A-IF-TSM-LI-S-NEL 2 99

hJAKl I-D--K-N-C--SVSTH-A-KHY-A-IF-TSM-LI -S-NEM 299

cJAKl SPALTLPSGDDG.LGYEVQVSGTTGISWRRKPVPNILIVKDKTKSKKNKADKQSKKEMTK 357

mJAKl -RCHSND--NVL.YEVM-TGNLGIQWRQKPNV-PVEKEKNKLKRK-LEYNKHKKDD-RN- 358

hJAKl NWFHSNDG-NVLYYEVM-TGNLGIQWRHKPNV-SVEKEKNKLKRK-LEYNKDKKDE-KN- 3 5 9

cJAKl RKTVMTIFSDFFEITHIVIKESCATIYSQDNKTMELDLFYRDAALSFAALVDGYFRLTVD 417

mJAKl LREEWNN--Y-P-WS-NK--N-N-K-SSHEE-VS-A- 418

hJAKl IREEWNN--Y-P-WS-NK--K-N-K-SSHEE-VS-A- 419 cJAKl AHHYLCTEVAPSSWQNLENGCHGPICTEYAIHKLRQEGNEEGTYVLRWSCTDYNYIIMT 4 77 mJAKl -D-PLI-H-IQ-N-S-M-FDN-L-- 4 78 hJAKl -D-PLI-H-IQ-N-S-M-FDN-L-- 4 79 CJAKl WCIEMDLCESRPVPQYKNFQIETSPQGYRLYGTDTFRPTLKELLEHLQGQNLRTENLRF 53 7 mJAKl -T-F-KSE.VLGGQK-F-VQKGR-S-H-SMDHF-S-RD-MN--KK-I-D-IS- 53 7 hJAKl -T-F-KSEQVQGAQK-F-VQKGR-S-H-S-RSF-S-GD-MS--KK-I-D-IS- 53 9 »*** _ja **** CJAKl QPVLVGLGQPRKISNLLVMTRDREPDSQRQPQVSQLSFHRILKEEIVQGEHLGRGTRTNI 5 97

mJAKl VLKRCCQPK--E-A-KKAQEW.-PVYSM-D-KDLI-H- 596

hJAKl MLKRCCQPK- -E-A-KKAQEW

.

-PVYPM-D-KDLI-H- 598

**IIa** *** _Ilia *** _{***j_va***}

cJAKl YSGVLKLKSEDDDDMGGYSQEVKVILKVLGSGHRDISLAFFETASMMRQISHKHIALLYG 6 57 mJAKl -T-LDYKDEEGIAEEKK. . I-DPS-A-V-VY- 6 54 hJAKl -Y-MDYKD-EGTSEEKK. . I-DPS-A-V-VY- 6 56 ****_**Va****** _*****yj;a*****+ cJAKl VCVRHQENIMVEEFVQYGPLDLFMRRQSIPLSTAWKFQVAKQLAGALSYLEDKKLVHGNV 717 mJAKl -DV-EG-H-K-DA-T-P-K-S-D- 714 hJAKl -DV-EG-H-K-DV-T-P-K-S-D- 716 ++**Viia**** * _*VIIIa** *** cJAKl CSKNILVARDGLDGEGGPFIKLSDPGIPITVLSREECVDRLPWIAPECVQDTANLSIAAD 777

mJAKl -T--L-L--E-I-SDI-VS--T-Q--IE-I-E-SK-V- 774

hJAKl -T--L-L--E-I-S-C-IT-Q--IE-I-E-SK---V--- 776 *j_Xa**** ***** _xa *** _***xia*** cJAKl KWGFGTTLWEICYNGEIPLKDKKLTEKERFYAAQCQLASPDCEELAKLMTHCMTYDPRQR 837 mJAKl --S-T-I-ESR-RPVT-S-K-D-R--N-N-- 834 hJAKl --S-T-I-ESR-RPVT-S-K-D-R--N-N-- 836 < i i to ********** _j **** CJAKl LFFRAIVRDIDMVEKQNPSIQP...VPMLEVDPTVFEKRFLKKIRDLGEGHFGKVELCRY 8 94 mJAKl P-M-NKL-E-D-VS.EKQPTT-H-R- 8 93 hJAKl P-M-NKL-E-D-VSRKKNQPT-H-R- 896

** ***ij**** **III* * _**IV**** ***

cJAKl DPRGDRTGELVAVKSLKPENREEQSSNLWREIHILRELYHENIVKYKGIWHEEGGRSIKL 954 mJAKl --E--N-Q-SGGNHIAD-KK--E-N-CM-D--NG- 953 hJAKl --E--N-Q-SGGNHIAD-KK--E-N-CM-D--NG- 956 ***** _v ******* *** * ** _VI ********* CJAKl IMEFLPAGSLKEYLPRNKAHIDLKTLLNYAVQICQGMDLLASRNYIHRDLAARNVLVENE 1014 mJAKl -S-K--NK-N--QQ-K--I-K-Y-G--Q-V-S- 1013 hJAKl -S-K--NK-N--QQ-K--V-K-Y-G--Q-V-S- 1016 ***YII**** ***YIII***** ***jx***** CJAKl NTVKIGDFGLTKSIKDNEGYYTVKDDLDSPVFWYAPECLIHCKFYRASDVWSFGVTMYEL 1074 mJAKl HQ-A-ETDKE-R-Q-I-LH-- 1073 hJAKl HQAETDKERMQSILH -1076 * _*x**** _**xi** CJAKl LTYCDISCSPMSVFLTMIGPTHGQMTVTRLVKVLEEGKRLPKPDGCSDRLYCLMRRCWEA 1134 mJAKl -SDF-AL--K-T-K-C-PN-P-EV-Q-K-F 113 3 hJAKl -SDS-AL--K--.NT-K-C-PN-P-EV-Q-K-F 113 6 cJAKl TPEKRIDFKGLIANFQQMIDNQ 1156 mJAKl Q-SN-TT-QN--EG-EALLK 1153 hJAKl Q-SN-TS-QN--EG-EALLK 1156

(6)

582 JH2 847 875 JH1 1156 c-JAKl

C-JH2

c-JHl

c-JH(l +2)

FIG. 3. Schematic

_diagrams

of

_full-length

and the various truncated forms of carp JAKl constructs. Thekinase-like and kinase domainsare

represented by horizontally striped

and dark

box,

_{respectively.}

Truncated moleculeswereconstructedas de-scribed in thetext.

normalize the

_samples

for differencesintransfection

_efficiency.

CAT and

_/3-Gal

activities in the extracts were measured

ac-cording

tothe

_{previous procedures}

(Herbomel

et

al,

1984).

The

acetylated

products

of the CAT assaywere

_{separated by}

thin-layer chromatography developed

with chloroform-methanol

(95:5, vol/vol),

visualized

_{by autoradiography}

and

_quantitated

by using

a

Phospholmager (Bio-Imaging

Analyzer

BAS

2000,

Fuji, Japan).

RESULTS

Isolation

_of

carp

JAKl cDNA

We

_amplified

carp brain cDNA

by

PCR

using

degenerate

primers,

an

approach

showntobeeffective in

_isolating

human and murine JAKl gene

(Wilks

et

al, 1991;

_Yang

et

al,

1993).

sets. PCR

_products

were then cloned into

_{polylinker regions}

of the

_reporter

vector

_{pCAT-Basic (Promega).}

Clone

_pJPl

contains the

_{flanking region}

_fragment

JP1,

nucleotides

—2,541

to +59. JP1 was

synthesized using

PCR

_primers

5'

-TTG

AAGÇTTTCCTCCTAGG

ATC AGG-C AG A-3'

(nucleotides

-2,541

to

_-2,522)

and 5'-GGGTCTAGAT-GCTTCAGTCGT-CATGATCAA-3'

_(nucleotides

+39 to

+59)

onthe 3-kb Sal I

fragment.

The

oligonucleotide primers

have additional sequences of Hind III and Xba I sites ateach

end,

respectively.

Fragment

JP1 was

purified

from the

gel,

di-gested

with Hind III and Xba

I,

and then cloned intothesame sites of

_pCAT-Basic.

Clone

_{spJP2, pJP3, pJP4, pJP5,}

and

_pJP6

were

similarly

_{constructed, except}

that the JP1

fragment

was

replaced

with JP2

(nucleotides

-2,541

to

-181),

JP3

(nu-cleotides

-2,541

to

-901),

JP4

(nucleotides -1,023

to

_+59),

JP5

(nucleotides

-528to

_+59),

and JP6

_(nucleotides

-181 to

+59),

respectively.

The

_pRSV-CAT

(Gorman

et

al,

1983)

was usedas a

positive

control.

Transfection,

CAT,

and

_{ß-galactosidase}

assay

Carp

fin

_epitheloid

cells,

CF

(Chen

and

Kou,

1986),

were maintained in Leibovitz's L-15 medium

_supplemented

with 10% fetal calfserum

(FCS)

at27°C. Subconfluent cultures

(ap-proximately

60%

confluent,

24 hr after

_plating)

in60-mm

cul-turedishes werewashed twice with Leibovitz's L-15

medium,

and incubated with

_{DNA-Lipofectamine complexes containing}

20 pg of the different CATconstructs

_together

_{with 5 pg of}

pSV-/3-galactosidase (ß-GAP)

vector

_(Promega)

in

_duplicate.

Transfection was carried out for 5

hr,

and cellswere washed with fresh Leibovitz's L-15 medium and fed with the same medium

_supplemented

with 10% FCS. After 40

hr,

cellswere

harvested,

washed in

_{phosphate-buffered}

saline

(PBS),

and

lysed

with 25 mMTris

_{phosphate pH}

7.8,

containing

2 mM

dithiothreitol,

2 mM

EDTA,

10%

_glycerol,

and 1% Triton X-100 at room

_temperature

for30 min. The total

lysates

were

scraped

fromthe dish and transferredto

_{microcentrifuge}

tubes. Cell debriswasremoved

by centrifugation

and theextractswere frozenat

—

70°C untiluse.Protein concentrationwasmeasured

by

the Bio-Rad

_protein

concentration

_quick-assay

method

(Bio-Rad, Richmond,

CA).

The

_pSV-ß-Gal

vector

_(Promega)

carry-ing

the SV40

_promoter

linked tothe

_/3-Gal

gene wasused to

¿w»

kDa M 1 94 75 45 28 22 17

ML

— C-JH1/JH2

#vvs

M c-JHl

B

kDa 94 75 45 28 22 17 anti-PY

FIG. 4. Demonstration of

_tyrosine

kinase

_activity

of c-JHl in E. coli. Strain JM109 cellsweretransformed with

pQE30

(lane

1),

pQEJH2

(lane 2),

and

_pQEJHl

_{(lane 3),}

_{respectively.}

After induction with 1 mM IPTG for 3

hr,

proteins

were

_prepared

and

separated by

NaDodS04-PAGE

and stained with Coomassie blue

(A)

orimmunoblotted with anti-PY mAb

_(B).

Positions of c-JHl and c-JH2 are shown on the

_right.

Lane

M,

Prestained molecularmassmarkers.

(7)

JAKl

Amplified fragments

ofabout220

_bp

were

purified

and

ligated

into

_{pGEM-T (Promega). Among}

the 20clones

_sequenced,

15 clones contained a

single

JAK-related sequence.

Using

this DNA

_fragment

as a

probe,

we screened an

oligo(dT)-primed

carp liver cDNA

library.

Schematics of thetwo

_{representative}

carp liver JAKl clonesobtainedfrom thisscreen areshown in

Fig.

1. On the basis ofDNA_sequence

analysis,

thesetwo

clones,

pJl

1 and

_pJ21,

areidentical_exceptthat

pJl

1 is0.7 kb whereas

pJ21

is 2.1 kb in

length.

It was found that

pJ21

encodes the carp

homolog

of mammalian

JAKl,

containing

residues from

465 to

1,156.

To obtain the

_full-length

cDNA

clone,

a

_300-bp

DNA

_fragment

from the 5' end of

_pJ21

wasusedas a

_probe

to

screen a_{carp brain cDNA}

library.

This

screening yielded pJ9

(1.9

kb),

asshown in

Fig.

1.

Therefore,

the

full-length

_carp

JAKl cDNA sequencesarederived from the two

_overlapping

clones,

pJ9

and

_pJ21,

and containa5'-untranslated

region

of 186

nucleotides,

a

coding

region

of

3,468 nucleotides,

anda

3'-untranslated

_region

of65nucleotides. The total

length

of carp JAKl cDNA is

3,719

nucleotides.

(The

sequence has been

de-posited

in GenBank withanaccession number

L24895.)

Structure and the

activity

of

carp

JAKl cDNA

The

_complete

_sequenceof carp JAKl cDNA encodesan_open

reading

frame of

1,156

amino acid residues witha

predicted

mol-ecularmassof 129 kD and the

protein

wastermed carp JAKl.

Allmembers of the JAK

family

haveseven

homologous

domains in the molecule that have been namedasJHsorJAK

_homology

domains. JH1 is a

carboxy-terminal kinase-catalytic

domain whereas JH2 isakinase-like

domain;

the other five JHsare

pre-sentin the farammo-terminal

_part.

Aminoacid_sequence

com-parison

of carp JAKl with human and murine JAKl

(Wilks

et

al, 1991;

Yang

et

al,

1993)

is shown in

Fig.

2. There isa

_higher

sequence

homology

in both JH1 and JH2

(70%

identity).

The overall sequence

identity

between carp and

human/murine

identity

between carp JAKl and murine

JAK2,

murine

JAK3,

and humanTYK2is

35%, 31%,

and

42%,

respectively.

Totestwhether the JH1or_{JH2 domains possess}

kinase-cat-alytic activity,

we

generated His-tag

fusion

proteins

of the JH2 domain

(c-JH2)

and theJH1 domain

(c-JHl)

of_{carp JAKl}

_(Fig.

3),

and

_expressed

thesefusion

_proteins

in E.coli

_(Fig.

4A).

The

tyrosine

kinase

_{activity resulting}

from each fusion

_protein

can be detected

_{by anti-phosphotyrosine}

monoclonal antibodies

(anti-PY mAb).

Duetothe lack of

_{endogenous tyrosine}

kinases in E.

coli,

there is littleornocross-reactive

background

for the anti-PY Western blot

_(Fig.

4B,

lane

1).

Among

the

_expressed

fusion

_{proteins (Fig.}

4A),

only

c-JHl

_{displayed tyrosine}

kinase

activity

and several

_{tyrosine-phosphorylated}

bands

_including

a

band

_{corresponding}

toc-JHl weredetected

(Fig.

4B,

lane

3).

By

contrast, C-JH2

(lane

2)

didnot_{express any observable}

ty-rosinekinase

_activity.

These resultsareconsistentwithan

ear-lier

_report

(Wilks

et

al, 1991)

andsuggestthat

only

JH1 isa

functionally

active kinase domain. c-JHl and C-JH2were fur-ther

purified by Ni-agarose affinity chromatography

and used

togenerate

polyclonal

antibodies

(see

below).

Full-length

and various

truncated

_{forms of}

carp

JAKl

are

_produced

in insect

cells

using

the

baculovirus

_system

Figure

5,

A and

B,

show that

c-JAKl,

c-JH(l

+

_2),

c-JHl,

and C-JH2were

_present

inboth the soluble and

Triton-A kDa 94 75 45 . 28 22 . 17 B kDa

VN^?V

„(*»»

sS» vN°'

JB^WW*1

10

„KcVO^

( anti-JH2 S&

_„ce«»

3ft0+ï>

..mJ**1

-« c-JAKl ., c-JHO+2) -« pp60 -. _c-JHl c kDa anti-JHl vf* 10 — c-JAKl — c-JH(l+2) -. pp60 — c-JHl anti-PY

FIG.5.

_Expression

of

_full-length

and various truncated forms

ofcarp JAKl in thebaculovirus system.Sf9 cellswereinfected

with

_wild-type

baculovirus,

AcMNPV

(lanes

1 and

₂₎

and a

variety

of recombinantbaculovirus

_{(lanes 3-10),}

_{respectively.}

After 60

hr,

cells wereharvested and

_separated

into Triton-soluble

(lanes

1, 3, 5, 7,

and

9)

andTriton-insoluble fractions

(lanes

2, 4, 6, 8,

and

10).

Both fractionswerefurther boundto

Ni-agarose gels

asdescribedin thetextand

_equivalent

amounts

ofbound

_patients

were

_separated

by NaDodS04-PAGE,

trans-ferredto

_{nitrocellulose,}

and immunoblotted with

_polyclonal

an-tibodies

_specific

for JH2 domain

(anti-JH2;

A),

for JH1 domain

(anti-JHl; B),

and a mAb for

_{phosphotyrosine}

(anti-PY; C).

Positions of

_prestained

molecularmassstandardsand their

(8)

insoluble fractionsas revealed

by

antibodies

_specific

for JH2

(Fig.

5A)

and for JH1

_(Fig.

5B).

Again,

the anti-PYmAbswere usedtodetect the

_tyrosine

kinase

_activity

of eachfusion

pro-tein. As a

control,

Sf9 cells were infected

_by

wild-type

bac-ulovirus and there was little orno cross-reactive

_background

for the anti-PY Western blot

_(Fig.

5C,

lanes1 and

2).

As shown in

_Fig.

5C

(lanes

3 and

4),

c-JH2 didnot

_{display tyrosine}

ki-nase

_activity.

Other fusion

_proteins,

c-JAKl,

c-JH(l

+

2),

and c-JHl all

_{expressed tyrosine}

kinase

_{activity (Fig.}

5C,

lanes

5-10)

and

_{autophosphorylation by}

themselves seemedtooccur as we

compared

their

electrophoretic

mobilities in

Fig.

5,

A and

B,

and the

_{corresponding}

mobilities in the anti-PY Western blot

(Fig.

5C).

Interestingly,

in cells

_expressing

c-JAKl and

c-JH(1

+

_2),

anadditional

_protein

of60 kDwasdetected

_by

anti-JHl antibodies and anti-PY mAb

_(Fig.

5B,

lanes

7-10,

and

_Fig.

5C,

lanes

7-10).

Moreover,

there were more

tyrosine-phos-phorylated proteins

bound to

_Ni-agarose

in the Triton-insolu-ble fraction of insect cells

_expressing

c-JAKl and

c-JH(l

+

₂₎

(Fig.

5C,

lanes 8 and

10)

than cells

_expressing

c-JHl. The pro-teins

_analyzed

werefractions boundto

_{Ni-agarose, presumably}

viathe

_His-tag

of recombinant

_proteins.

Therefore,

they

were either

_{cleavage products}

of JAKl related

_proteins

orassociated

proteins.

c-JHl and C-JH2

interact

with each other

To demonstrate the association of c-JHl and

c-JH2,

anti-JHl

antibody

was used to

_precipitate

c-JHl from the extract of vAcJHl- and vAcJH2-co-infected

cells,

and the associated c-JH2wasdetected

by blotting

withanti-JH2

antibody.

As shown in

_Fig.

6A,

C-JH2 is

_{co-precipitated by}

c-JHl.

_Similarly,

c-JHl

is also

_{co-precipitated}

by

c-JH2

_(Fig.

_6B).

_{Because both}

pro-teins are

highly expressed

under these

conditions,

there is the

possibility

that this interaction is somehow

_nonspecific.

Therefore,

another recombinant baculovirus

vAcCAT,

which carries the

_{chloramphenicol acetyltransferase}

(CAT)

gene un-der the control of the same

_polyhderin

_promoter,

was usedto coinfect with vAcJHl or vAcJH2. The

immunoprecipitation

data showed that neither anti-JHl noranti-JH2

antibody

was ableto

_precipitate

CAT

_protein

from thecoinfected cellextract

(data

not

_shown).

Moreover,

neither anti-JH2noranti-JHl

an-tibody

wasableto

_precipitate

c-JHlorC-JH2 from theextracts

from cellsinfectedwith vAcJHl orvAcJH2 aloneasshown in

Fig.

6

(lane 1).

Therefore,

C-JH2seemstointeract

_specifically

with c-JHl and

_{possibly by}

_{tyrosine-phosphorylated by}

c-JHl

(see

texts

_below).

JH2 domain

is

_{tyrosine-phosphorylated}

_by

c-JAKl and

c-JH(l +2)

Because c-JHl and C-JH2were associated with each other

(Fig.

6),

we

_sought

to

_investigate

whether c-JH2 isasubstrate forc-JH1

_by

co-infection

_experiments

in which insect cellswere co-infected with vAcJH2 and vAcJAKl or vAcJH2 and

vAcJH(l

+

_2),

instead of co-infection with vAcJH2 and vAcJHl. This avoided

_ambiguity

indata

_{interpretation}

due to

the similar

_mobility

of c-JHl andC-JH2on

NaDodS04-PAGE.

The

_{experimental procedures}

werethesame asthose described in the vAcJHl and vAcJH2 co-infection

_experiment.

Proteins boundto

_{Ni-agarose gels}

were

_{analyzed by blotting}

with either anti-JH2

_antibody

oranti-PY mAb. The control

experiment

was carried out

_{by infecting}

insect cells with eithervAcJAKl or

##

B

kDa

94

-75 -45

-28

-22 -17

-IP

F

_{& &}

1 2 3

#^

ir jo w *

_<§

_*

Blot

:

_anti-JH2

kDa

94

-75 -45 -28 -22 -17

-IP:

9

Cv ¡y » « S

S¡ S

Blot

:

_anti-JHl

FIG. 6. Association ofc-JHl with C-JH2. Sf9cells wereinfectedwith vAcJH2 alone

(a,

lane

1),

vAcJHl alone

(B,

lane

1),

and co-infected with vAcJHl and vAcJH2

(lanes

2and

3),

respectively.

TheTriton-soluble fractionswere

immunoprecipitated

with normal

_{guinea pig}

antiserum

_(pre-imm;

Iane2),

anti-JHl antibodies

(A,

lanes 1 and

3)

and anti-JH2 antibodies

(B,

lanes 1 and

3).

The associated

_proteins

were

_{analyzed by}

NaDodS04-PAGE,

transferredto

nitrocellulose,

and immunoblotted with anti-JFK

(A)

oranti-JHl antibodies

(B).

Arrowheadindicates

_positions

of c-JHl and

C-JH2,

respectively.

(9)

JAKl

vAcJH(l

+

₂₎

alone. As shown in

_Fig.

7B,

C-JH2indeedwas

tyrosine-phosphorylated

by

c-JAKl

(lane 2)

and

_by

c-JH(l

+

2)

(lane 4).

This suggests that

_{transphosphorylation}

of C-JH2

by

c-JHl mayoccurwhen

_they

are

expressed

in

high

levels

to-gether.

RT-PCR

_of

brain and liver mRNA

Asdescribed

above,

two

_overlapping

_clones,

_pJ9

and

_pJ21,

wereisolated from different tissues. To

_investigate

whether

tis-sue-specific

alternative

_splicing

occursandto

_analyze

the un-cloned

_regions

inthetwo

tissues,

fivesets of

_{specific primers}

wereusedto

_perform

RT-PCRonmRNAderived from

com-4.4 kb—

2.3 kb

_

2.0kb—

0.6 kb—

*

if

<o

&

& .

# %

Q %

&

BLBLBL BL BL kDa 94 _ 75 -45 _ 28 _ 22 _ 17

-B

kDa 94 . 75 -45 . 28 -22 -17

c#

<^.

V

Ä^>*>

MF

»i* tr

_<fif?

- c-JAKl <—— ^

_c-JH(l+2)

anti-JH2

Hi

&

^c^>:>

_4*"

_4P

MF

-, c-JAKl • -«

c-JH(l+2)

-*

pp60

» -« C-JH2 anti-PY

FIG. 7.

_{Transphosphorylation}

ofC-JH2

_by

c-JAKl and

c-JH(1

+

_2).

Sf9 cellswereinfectedwith vAcJAKl alone

(lane

1),

both vAcJAKl and vAcJH2

(lane

2), vAcJH(l

+

₂₎

alone

(lane 3),

and both

vAcJH(l

+

₂₎

and vAcJH2

(lane 4),

respec-tively.

The Triton-insoluble fractions were

_prepared

and then incubated with

_Ni-agarose

gels

and

_equivalent

amounts of bound

_proteins

were

separated by

NaDodS04-PAGE,

trans-ferredto

_{nitrocellulose,}

andimmunoblottedwith JH2 anti-bodies

(A)

or anti-PY mAb

(B).

Prestained molecular mass markers

(in kilodaltons)

areshownonthe left.

FIG. 8. RT-PCR

_analysis

of_{the carp JAKl kinase}

_transcripts.

mRNAs derived from brain

(B)

and liver

(L)

tissues were

primed

with

_oligo(dT)

and random

_primers

and

_subjected

to re-verse

transcription.

The

_resulting

cDNAwas

_amplified

with five

primer

sets,

i.e., F3/R3,

R4/R2,

F3/R2,

Fl/Rl,

and

F1/R2,

and thePCR

_products

were

analyzed

_{by electrophoresis}

on a 1% agarose

gel.

The sequence of each

primer

wasdescribed in the

DNAConstructionsection.

mon_{carp brain and liver tissues. PCR}

_products

_{from both} tis-sues were identical in

_{length (Fig.}

8).

Only

the uncloned

re-gions

inthetwo

_tissues,

_i.e.,

PCR

_products

from

_primers

F3/R3

and

_{F4/R2 (from brain),}

and the DNA

_fragment

from

Fl/Rl

(from

liver)

were

_sequenced.

_{All of the nucleotide sequences} werethesame as thosewe

_deposited

in GenBank withan ac-cession number L24895. These sequences,aswellasthose from two

_overlapping

clones,

_pJ9

and

_pJ21,

suggestthatatleasttwo

identical

_transcripts

_{from carp brain and liver tissues encode the} same

full-length

_{carp JAKl kinase.}

Genomic

organization

_of

the

carp

JAKl

gene

Asaninitial

_step

to

_investigate

the

_regulation

of carp JAKl gene

expression,

we also _{cloned and characterized the carp} JAKl_gene. Four

_{positive phage}

clones,

termed Jl to

_J4,

were

isolated from a

_Stratagene

_{carp liver}

_{genomic library}

_with a

DIG-labeled

_full-length

_{carp JAKl cDNA}as a

probe.

We are unable to_{fill the gap between}

_phage

cloneJl and J2

_by

PCR

amplification

of

_genomic

DNA with a setof

_{oligonucleotides}

that

_correspond

to one end of clone Jl andthe other end of cloneJ2.To locate allexons,these

_phage

cloneswere

_analyzed

by

Southern

_{blotting, subcloning,}

and

_sequencing.

As shown in

Fig.

9,

the restriction map of each

genomic

clone was

con-structed

_{by digesting}

the

_phage

DNA with a

_panel

of restric-tion enzymes

separately

orin variouscombinations:

5a/1,

Bgl

n,

Hind

III,

Xho

I,

and Eco RI. On thebasis of the nucleotide sequences of subcloned

fragments,

the carp JAKl gene is

com-posed

of24exonsthat spans atleast 31 kb ofDNA

_(Fig.

9).

The_sequencesaroundthe exon-intron boundarieswere deter-mined andareshown in Table 1. Allexon-intron boundaries

identifiedconformedtothe

GT/AG

splice donor/acceptor

rule

(Breathnach

et

al,

1978).

Some exons were

_relatively

small

(88-108

_bp),

whereas the first and the sixth exons were

_large

(237

bp

and 349

_bp).

The size ofintronsvaried

_{considerably,}

ranging

from >3 kb

(intron

1)

to 100

_bp

(intron 19).

The first exoncontainedthe5'untranslated

region,

andthe secondexon

(10)

exon untranslatedexon

I_L

J_L

i i

2S

_o— CJ00 Wffl

_u_

> Di o -Ü CO — _-, Sx m a

_LL

J_L

> oí

Exon

no l 2 3 4 5 6 7 8 9 10 11 1213 14

JAK,

^JIJUL/IIII

IJUJU1JLJUI

15 1617181920 21 22 23 24 2.5kb >3kb 1.8kb lkb 1.2kb 1.5kb 2.3kb 2.2kb 1.5kb 1.8kb 1.5kb 1.4kb

Jl

/A

J2

J3

J4

_"//

FIG. 9. Genomic

_organization

of the carp JAKl gene. Exons are indicated

by

boxes numbered from 1 to24.Solid boxes

in-dicate_{the carp JAKl}

_{coding region}

whereas open boxes

represent

the 5' and3' untranslated

_regions.

Intronsand the5'-and

3'-flanking regions

are indicated

by

the solid lines. The entire gene spans atleast 31 kb in

length

andcontains 24exons. A re-striction mapwasshown above the

_genomic

structure.Three

_{overlapping phage}

clones,

J2-J4 andone

_{nonoverlapping}

clone, Jl,

were isolated froma_{Lambda FIXII carp}

genomic library.

Agap between

phage

clones Jl and J2was notobtainable

_by

PCR

amplification

of

_genomic

DNA.

containedthe

_putative

translation initiation site. The

largest

in-tron

(>3 kb)

_separates

exons1 and 2. The JH2 domainwas lo-catedon exons 11-17 and the

_catalytic

JH1 domainwaslocated on exons 18-24. Exon 24containedthe last 33 amino acidsas

wellas the 3' untranslated

region.

The

_promoter

and

exon/in-tron_{sequences of carp JAKl kinase gene have been}

_deposited

in GenBank with 10 serial accession

numbers,

from U53685to U53694.

Table 1. Exon-Intron Organizationof theCarp JakI Gene

exon exon size

number

_(bp)

3'end of

5'end of

_approximate

3'end of

5'end of

amino

acid

the

exon

the intron

size

(bp)

the intron

the

exon

interrupted

1 242 TGC CTG ACG AG gtaaggacga >3000 tatcctgcag T GTC TGG ATG

2 208 CAA GAA GAT GCC gtaagcgaga 1800 ttttccccag ACA TCT CAC Ala67 (3)

3 125 TAC CGT ATG AG gtgagtgggc 1000 gtttacacag G TTT TAT TTT Argl09 (2)

4 154 CTG TTT GCA CAG gtgaggtaca 400 _tatgttttag GGC CAG TAT Glnl60 (3)

5 164 GCTGAG ACC AG gtgaggtaca 148 atctttgtag C TAC AAG CGA Ser215 (2)

6 349 AAG CCT GTA CCG gtgagtttag 250 cttttgatag AAT ATT CTG Pro331 (3)

7 180 AAT AAA ACT ATG gtaatttcca 600 ttgcccacag GAG TTG GAC Met391 (3)

8 158 GGA CCT ATC TG gtatgaccat 1200 ttctctacag C ACA GAG TAT Cys444 (2)

9 115 GTC TGC AH GAG gtacacacac 1500 tctctctcag ATG GAC CTA Glu482 (3)

10 199 CAA CCC AGA A gtaccagcac 2300 tttccttcag AA ATT TCC AAC Lys549 (1)

11 110 GAG ATT GTA CAG gtgatatttt 900 ttttattcag GGT GAG CAT Gln585 (3)

12 150 GAT ATC TCT CTG gtaagatgca 187 ctcctctcag GCT TTC TTT _Leu635 (3)

13 89 CAT CAG GAG AA gtaagtacct 2200 _ctgtctgtag T ATC ATG GTG Asn665 (2)

14 127 CTC AGC TAT CTG gtaaagaaac 1500 aatcctgtag GAG GAC AAG Leu707 (3)

15 136 AGCAGA GAA G gttgagagat 820 gtgtctacag AG TGT GTG GAC Glu753 (1)

16 152 AAGCTC ACA GAG _gtaacagcat 111 ctctgagcag AAG GAG AGG Glu803 (3)

17 152 GAG AAA CAG AA gtatgaccta 457 ttctctccag T CCT TCC ATT Asn854 (2)

18 88 GAT CTTGGA GAG _gtattttctg 110 ctcttttcag GGT CAC TTT Glu883 (3)

19 193 CAC GAA GAA G gtaaagccac 100 _ttgtttcaag GT GGG AGA TCC Gly948 (1)

20 125 CAG ATC TGC CAG gtaacatcat 1800 ttaactccag GGC ATG GAC Gln989 (3)

21 173 CCA GTG TTCTG gtaagaatca 170 attttcccag G TAT GCC CCA Trpl047(2)

22 118 AGC CCT ATG TCG _gtaagtggcc 1500 aaccattcag GTG TTC CTT Serl086(3)

23 111 TGC TCA GAC AGG _gtaatatata 1400 gttttgacag TTG TAC TGT Argll23(3)

24 164 ATG AH GAC AAT CAG TAACTATGGAAATAAGATGAGATGCAGACTCCACCTCTTTTGTAAGAGGAAGTTCCAGAGAGACCAAAAAAAAAAA

_

(11)

JAKl

.NF-IL6 . -2541 TCCTCCTACGATCAGGCAGAAAATGTTTTnGACATTTATTTTGACATTATGTTGTTGTTTTGGGAAATAAAAAATCATTAAAATATTCACnnCACTC -2441 ATGTTTTTCCTGTAGACATCACGACTATAAAGAAAATTAAGACATCATTTCAGTTTGCAnTCATTTAATGTTnCTACAATTTCnTTCTTTCTGTGTC -2241 TTTTGGCACAGAAATATCCTTTTCAAATAGACACnGTAATCAAACTTTGCACAGACTTTAGTnCTGTGTTTnCAAAGACTACAAAATGTATATAAGT HNF-5. ... -2141 TAAAACAACAnGTACATCCTTATTTAnATGCATATnGTGTAAATATTAGCAGAAATGGGAAnCCATCCTTnACATATAnAACATTTGnTTAGG -2041 GCGACnACTACAAAAAGATGnTACnATATGAAACAATTTGACAACCAAACMATAGAAGTTTTAAAAAAAAAATAATACCTGCTTTTCTGTTCCCAT -1941 AAAACTGCTGAnTGTGATGTCAGACATnAAAAGCCATGAAGAGAAAGATATCATGGCCAAACCCCAAAACATTTGGAATCTCTGAAAAGCCCAATATG NF-IL6 .... -1741 CAGnCTAGGAGGnAATATAnGAATATCATTTTGTACAATATCAAAGCATATATGATGTATAAATTAGTTACTAAACAnnAnCAATTCATAGCAT -1641 nGGACACACACCATGAAGATnACAGTGTAGTATGATAAGTCnnCTGCCCATnCGTnATATAAnATAGACCATnTCCCATGATGTTAGTAAAT .API -1541 TAGACCCAGGTATTATCCTCTGTCACTTGCCCCnGnACATCAGCCCTnAACTCTTGAGTCATCTGAACATTTTAGAATCATATAGTAAGGTGAGTAA -1441 GTGGTCTGTAATGnGTGATATATGGAAATGCAGnATACAATnATTTTTTCnTTITnCAGAAAATAAAAATGTTTTCTCATCTGAAAAGCTGGGTC NF-IL6 . HNF-5 .... -1341 ATTnCAAnTGATGTTTACATGTATTTCGGAAATCCTCATCTAAATATTTGCTGAATnCTGTCTTAGCTTTTAGATGAGTGTTTATGGCAGTAnGCA . API -1241 CGTAGACTCATCACGGACCAAnATAGCCCATGACTCATCCAACGATAGGAGTTTCCAACTGGACTTTTTCCCCCCAGTGTnGTTTAAATAnAnGTC -1141 AACATATCTCAACACCATATTTGACATAAATATGTAnGTTGCAAATCATTnGACATGTCTGACAACATTTATATGGAAATGTTGATAAGGATAGACAA -1041 AAATGTATTTAATAGTAGAATnGAACTGAACGTTnACTCTAAAAACCTATTAAACTCCAATACACACAGTCTAATAnGTTTATATATCTACCCGAAC -941 nCTGnGAACTTAnGATCAGTCACAnaCCCAATGCTCTGCAGCCCACAGTTTCTGGCAACTCATATGATGCTGAGGACGGAAGTCCCTCCCTTTCA -841 CGGCAGGCnAAACCTCATGTCnAAAGACCATCTGCGTAATCAGAGAGGGGTTTGTGTTnGCTGATACCTATTTTAAAAACAGACACGTACCTGTAAA E2A ... -741 GCGGAAACAGCTGATTGATTMCTTAATGTnAGGAnAGGACTTrATGGACAAAAATAAACAACAGTAATAAAATGTAACAGnCAnAATAATATTTC NF-IL6 . . GHF-5 ... -641 TATTTACATTTTAnTCTGAAATCTAAAnATnATGAnATATCCAnATnATGAATCTGCAAMCATCTTTTACAGTGnGTGCTGTTACTGTTTAT -541 ATAGACTAGATAACTGCAGTATCTAAGCGnACnTTAAATGAGAAGCATAATAATGCnAGnAGCAGTAAnATTTAATATATGAATCAAnATTAAT -441 GGTTGTTATATnAAAGTGGTCATGTGGCATACCAnCAATGGACAAAATTnATCCGAGATATCTTTnAATACTACATATTTAACGAAGTGAAATCAG . TATA-box. -341 CGCnAAGGATCnCATGTAnATGAnCTAATCAnTTCAAACCATTTGAATCAnGGAGAGAGAGAAAAAAAAATGATATGTATAAATATGATGTTiT NF-IL6 ... -241 GGGAAATTTATGCATTTATGTTTACATTACAGCAATGCACTCTAAGCGAGAGTTTTGATCGCATGCATAAATAGTAAnCGTCTTTTAACTATATACAGT -141 ACAGAGGCAATGCGTCCAGTAGCCACAACAAAAAATCGCTGGCTTACTCTCTGCnAAGGGnAAAGCCAnGGTACAGAATTTGAnGACAGCCGCATG . CCAAT ... .+1 .... . -41 AGCCAATAAGCGGCATGnACAGACCTCGTCGnACCCTATTnCGGGAATGTGTTCGACGCTTGACAGGAAAGGTTAGTTGATCATGACGACTGAAGCA

.*

_{( transcription}

start site of carp JAKl gene

)

.

+60 CGGnTGTAGCACTCTGAAGTGAAGTnGTnTGAAAATAAACCTCGTGGAATAAACCAnATACTATCGGACAnAnnGGAGTGAAATAACGAGCAC

+250 +160 TTTTACATCAACAAAAAACCAAGAGGAAGTGTTCAAACGTCTGTAATGATCGAAAnAAACGGAAGTAGCTTTGCCTGACGAGTGTCTGGATGCCAGAAC

(

translation start site

)

FIG. 10. Nucleotide_{sequence of the}

5'-flanking region

of the carp JAKl gene. A 28-mer antisense

oligonucleotide

usedfor

primer

extension

_analysis

is underlined. The candidate

transcription

startsite

_{by primer}

extension

(see

Fig.

11)

is indicated with anucleotidenumber

(+1)

andan

_asterisk,

which is locatedat249

_bp

_upstream

tothetranslationstartsite.Potential

_binding

sites fora

variety

of

transcription

factors arealso marked and underlined. The

_promoter

andexon _{1 sequences of carp JAKl kinase} gene has been

deposited

in

GenBank,

accessionnumber U53685.

Determination

of

the

_{transcription}

initiation site

The

_{transcription}

startsitewasdetermined

by

primer

exten-sion

_{analysis using}

_poly(A)+RNA

from carp liver. We useda 28-mer

_{oligonucleotide}

labeled with

32P

atthe5' end. The

ex-act

_position

of the extended

_product

wasdetermined

by

align-ing

the

_sequencing

ladderobtainedwith thesame

_primer.

One

major

extended

_product

wasrevealed and

corresponded

tothe siteat249

_bp

_upstream

tothe initiatormethionine codon. For

describing

the

_{5'-flanking region}

of the carpJAKl _gene, we