Molecular cloning and chromosomal mapping of the human gene for the testis-specific catalytic subunit of calmodulin-dependent protein phosphatase (calcineurin A)

Vol.

188,

No.

October

15.

1, 1992

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Pages

1992

265-271

MOLECULAR CLONING AND CHROMOSOMAL MAPPING OF THE HUMAN GENE FOR THE TESTIS-SPECIFIC CATALYTIC SUBUNIT OF CALMODULINDEPENDENT PROTEIN PHOSPHATASE (CALCINEURIN A) Taro

Muramatsu

and

Randall

L. Kincaid

Section on Immunology, Laboratory of Molecular and National Institute on Alcohol Abuse and Alcoholism, Received

August

28,

Cellular Neurobiology, Rockville,M D 20852

1992

SUMHARY: A cDNA for an alternatively spliced variant of the testis-specific catalytic subunit of calmodulin dependent protein phosphatase (CaM-PrP) was cloned from a human testis library. The nucleotide sequence of 2134 base pairs (bp) encodes a protein of 502 amino acids (Mr ~57,132) and p1 7.0. The cDNA sequence differs from the murine form of this gene by a 30 bp deletion in the coding region, the position of which matches those in the two other genes for the catalytic subunit. These data indicate that this alternative splicing event arose prior to the divergence of the three genes. The deduced sequence of the human protein is only 88% identical to the homologous murine form, in striking contrast to the other two CaM-PrP catalytic subunits which are highly conserved between mouse and human ("99%); this indicates a more rapid rate of evolution for the testis-specific gene. Analysis of Southern blots containing DNA from humanhamster somatic cell hybrids show that the gene is on human chromosome 8.

Calmodulin-dependent involved

in a wide

modifier

of

induced

glycogen

form

of

of

that

to

this

kDa, or

"ol(

variants and rats,

and

"5"

are

through

in mammals forms)

known

and are

are

(8,9,14). highly

acting

broad-based

may alter

CAMP regulated mechanisms

three (7-13).

highly

These conserved

genes

(5) (6).

three

in brain have

been

species

it

phosphorylation

have

been

genes and

seems here

as

(60 and 18

cloned

(PPZBal

sperm

and a unique

Thus,

subunits

CAMP

through

of the

and regulatory genes

to

activity

in muscle.

distinct

between

opposition

than

Two of the expressed

as epinephrine-

the motility

flagellum

different of catalytic

in

phosphorylation the

to be

as a Ca"-dependent

phosphatase

In testis,

with

appears

such

may act

by CAMP-dependent

To date,

subunit

it

of PPl (4).

be associated

calcineurin)

In some cases, (3),

by initiating

is composed

respectively).

(PP2B,

activities,

(1,2).

muscle

phosphatase

perhaps

The holoenzyme catalytic

in

be controlled

although

biological

inhibitors

PP2B appears

phosphatase

status

cascades to

plausible well,

of

breakdown

dephosphorylation thought

spectrum

phosphorylation

phosphorylation is

protein

for

the

and PPZBaZ,

alternatively-spliced

identified

in humans,

(99% amino

acid

mice

identity).

0006-291X/92

$4.00

Vol.

In contrast,

expression

specific,

and

regulated of

BIOCHEMICAL

188, No. 1, 1992

has

of

been

cells.

This

paper form

(PP2Bar3,

only

showing

alternatively-spliced human chromosome

gene

reported

developmentally,

germ

a third

AND BIOPHYSICAL

mice

an apparent

reports of

in the

human

RESEARCH COMMUNICATIONS

or "y")

is essentially

(10).

The

correspondence cloning

PP2fW

and

testis-

testis with

isoform thematuration

characterization

and documents

its

is of

an

localization

on

8.

HATERIALS

AND HETHODS

Materials: Restriction endonucleases, the Klenow fragment of DNA polymerase I, T4 DNA ligase and reagents for DNA sequencing, using a modified form of T7 DNA polymerase (Sequenase) were from United States Biochemical. All components used for polymerase chain reaction (PCR) were obtained from Perkin-Elmer/Cetus and oligonucleotide primers were synthesized with a Cyclone Plus DNA synthesizer (MilliGen/Biosearch, Novato, CA). Southern blots containing Eco RI-digested DNA from human-hamster hybrid cell lines were obtained from Bios (New Haven, CT), as were Hybond nylon membranes used for the screening of a phage library. Cloninq of the human CONA: Template for the hybridization probe was prepared by PCR amplification of the open reading frame (ORF) of a murine testis clone (10); after purification of the PCR fragment, it was biotinylated by using random primers as described (15). A human testis cDNA library constructed in lgtll (Clontech, Palo Alto, CA) was screened by plaque hybridization (8). After isolation of clones, phage DNA was purified from plate lysates by immunoaffinity procedures using LambdaSorb (Promega) and subcloned into pUC vectors as described DNA was prepared by the alkaline lysis method. DNA sequencing (8) - Plasmid reactions for the longest clone, using the dideoxy termination method (16), were done twice on both strands using primers located ~200 bp apart. Southern blot analysis: The template for the hybridization probe was generated by PCR amplification of a 122-bp region in the human cDNA (see Fig. 1). A radiolabeled hybridization probe was prepared by primer extension of the template using both sense and anti-sense primers, essentially as described (8). The Southern blots containing chromosomal DNA were prehybridized for 4 hrs at 420C in a solution containing 6x SSC, 5x Denhardt's reagent, 0.5% SDS, 100 pg/ml salmon sperm DNA and 50% formamide. Hybridization was carried out for 15 hrs at 42°C in the same solution containing 1~10~ cpm of probe per ml of hybridization solution. The blots were then washed sequentially for 15-min periods with 2X SSC/O. 1% SDS and 0.2X SSC/O. 1% SDS at room temperature, followed by a final I5 min wash at 37°C in 0.1X SSC/O.l% SDS.

Clonino

of

A human biotinylated of 40,000

testis

probe

(Fig. identity),

l),

but

cDNA

286

lacks is

except

testis

point

bp of

a region

highly that

5'

in

clones

for of

7.0.

were

30 base

to pairs

the are 266

found.

library:

screened

(a3 isoform) All

of molecular

using (10);

of these mass,

a out

contain

57,132,

that

The clone having the longest insert, region (UTR) and 339 bp of 3' UTR

of polyadenylation.

homologous

was

subunit

a protein

untranslated

a human testis

Lgt-11

catalytic

3 positive

1509 bp coding

isoelectric

possesses

constructed

the murine

screened,

ORF of

has a predicted

human

library

for

plaques

an identical HTa-1,

RESULTS AND DISCUSSION soliced PP2Bar3 cDNA from

an alternatively

murine deleted

The nucleotide testis-specific in

a region

sequence form between

the

of the (~85% CaM-

Vol.

188,

No.

1, 1992

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

-286

GGGCCACCCTTAECAG CGGTCCCCGTCGGTGCCGAAGCGGTG~TCC -241 CCGCCTTAGCCGCTGCGCCTCCCAAGAGAG CGGCCGGTGGGCCCTCGTCCTGTCAGTGGC GTCGGAGGCCGGCCTGCGGTGGCCGCGCCC TTCTGGTGCTCGGACACCGCTGAGGAGCCG -12, GGGCCGGCCACGGCTGCCTGACGGCTCCGG GCAGCTAAGGCTGCCCGAGGAGAAGGCGGC GGCCGCGGCGTAGGCGCACGTCCGGCGGGC TCCTGGAGCCTGGAGGAGGCCGAGGGGACC -1 ATGTCCGGGAGGCGCTTCCACCTCTCCACC ACCGACCGCGTCATCAMGCTGTCCCCTTT CCTCCAACCCAACGGCTTACTTTCAAGGAA GTATTTWGAATGGGAAACCTAAAGTTGAT 120 MSGRRFHLST TDRVIKAVPF PPTORLTFKE V F E W G K P K V 0 40 CTTTTAAAAAACCATTTGGTMGGAAGGACGACTGGAAGAGGAAGTAGCCTTAMGATA ATCAATGATGGGGCTGCCATCCTWCCUIGAGAAGACTATGATAGAAGTAGATGCTCCA 240 VLKNHLVKEG RLEEEVALKI INDGAAILRO E K T M I E V D A P 80 ATCACAGTATGTGGTWTATTCATGGACAA TTCTTTGACCTMTGMGTTATTTGMGTT GGAGGATCACCTAGTMCACACGCTACCTC TTTCTGGGTGACTATGTGGACAGAGGCTAT 360 ITVCGDIWGO FFDLMKLFEV GGSPSNTRYL FLGDYVDRGY 120 TTCACTATAGAGTGTGTGCTGTATTTATGG AGTTTAAAGATTMTCATCCWCATTG FSIECVLYLY SLKINHPKTL

TTTCTGCTTCGGGWTCATGMTGCAGG CATCTTACAGACTATTTCACCTTCAMCAG 480 FLLRGNNECR HLTDYFTFKO 160

GAATGTCGAATCAAATATTCGGAACAGGTG TATGATGCCTGTATGGAGACATTTGACTGT CTTCCTCTTGCTGCCCTCTTAAACCAGCAG TTTCTCTGTGTACATGGACGAATGTCACCT 600 YDACMETFDC LPLAALLNOO FLCVHGGNSP ECRIKYSEOV 200 GAAATTACTTCTTTAGATGACATTAGGAAA TTAGACAGGTTTACGGAACCTCCCGCCTTT GGACCTGTGTGTGACCTGCTTTGGTCTWT CCCTCAGAGGATTATGGCAATGAGAAGACC 7’20 EITSLDDIRK LDRFTEPPAF GPVCDLLUSD PSEDYGNEKT 240 TTGGAGCACTATACCCACMCACTGTCCGA GGGTGCTCTTATTTCTACAGTTACCCTGCA GTTTGTGAATTTTTGCAGMCMTAATTTACTATCMTTATCAGAGCCCATGAAGCCCAA 840 VCEFLOYNNL LSIIRAHEAO 280 LEHYTHNTVR GCSYFYSYPA GATGCTGGGTATCGAATGTACAGGAAGAGC CAAGCCACAGGCTTTCCATCACTTATTACA ATTTTCTCTGCCCCCAATTACCTAGATGTC TATAACAAYAkAGCTGCTGTGTTGAAATAT 960 OATGFPSLIT IFSAPNYLDV YNNKAAVLKY 320 DACYRNYRKS GAAAACAATGTCATGAATATCAGGCAGTTT MCTGTTCTCCACACCCCTACTGGCTTCCA MCTTTATGGATGTTTTCACATGGTCTTTG CCTTTTGTTGGGGAMAAGTCACAGAGATG 1080 ENNVINIROF NCSPHPYYLP NFNDVFTYSL PFVGEKVTEN 360 CTGGTAAATGTGCTCAACATATGCTCTGAT GACGAACTGATTTCTGATGATGAAGCAGAA GGAAGCACTACAGTTCGTMGGAGATCATC AGGAATMGATCAGAGCCATTGGGAAGATG 1200 GSTTVRKEII RNKIRAIGKM 400 LVNVLNICSD DELISDDEAE GCACGGGTCTTTTCMTTCTTCGGCMGAA AGTGACAGTGTGCTGACTCTCAAGGGCCTG ACTCCCACAGGCACACTCCCTCTGGGCGTC CTCTCAGGAGGCAAGCAGACTATCGAGACA 1320 440 ARVFSILROE SESVLTLKGL TPTGTLPLGV LSGGKOTIET GCCATCAGAGGGTTCTCGCTTCAGCACAAG ATCCGGAGTTTTGAAGAAGCGCGAGGTCTG GACCGAATTMTGAGCGAATGCCACCCCGA MGGATAGCATATACCCTGGTGGGCCAATG 1440 AIRGFSLONK IRSFEEARGL DRINERWPPR KDSIYPGGPM 480 AAATCTGTAACCTCAGCACACTCACATGCT GCGCACAGGAGCGACCAAGGGAAGAAAGCC CATTCATGACTTAGAGTCCTGCCGTGCTCA GGTGGATCTAAAACTCAAGAACAAATTCTA 1560 KSVTSAHSHA AHRSDOGKKA H S . 502 TTTATTTATTATTGGAAAATCGCMC TCAAAACMCTTCAACCTGGAGGTGCATTT ATMTTCAGTCTGCATTTATTCTGTW GGTGACTGTTTTATAAATTCTTTTMTTTTA 1680 TGTTCAATATATATAAAAAGTGCATCTGTT TTGTTTTTCCCTTTTTTCTCCATAATTTTAAGAAATGAATCTGATTGTTGTCMCACATT TGTGAAGTCTTGTGCTATAAAGGGGAACTT 1800 CCCCTAATAAAAGCGCCTTGGAAACCTCAA ACCTGGCTTTCTCACCCC 1848 5’UTR

3’UTR

286 b

ORF I SSPl

0

Fiqure

1.

400

Nucleotide

800

I N&l

1200

339 bp

I Xho2

1600

2000

amino acid sequences for the catalytic subunit The nucleotide and deduced amino acid sequences of the clone HTa-1 are presented, with positions in the 5' UTR represented as negativenumbers. The underlined region (bp 271-392) represents the segment that was PCR-amplified and used in preparing the hybridization probe for Fig. 3. A map of unique restriction endonuclease sites is shown at the bottom of the figure. ORF, open reading frame.

of Can-PrP

binding

(7)

and

1509 tp I Pstl

deduced

from human testis.

and autoinhibitorjdomains

amino

acid

440-449

shown

that

homsldgou‘s

in the

deduced

(17);

this

mouse sequence

gqternatively-spliced

forms 267

corresponds (Fig. exist

2).

to

the

Although for

the

neural

absence it

of

has been (al,

a2)

Vol.

BlOCHEMlCALANDBlOPHYSlCALRESEARCHCOMMUNlCATlONS

188, No. 1, 1992

Hum Mur

MSCRRFHLSTTDRVIKAVPFPPTPRLTFKEVFENGKPKVDLKIINDCAAILRP ..V..PQF...E...........R...L..........M.L.........-V.--.............K.

70 70

HUll Mur

EKTMIEVDAPlTVCGDIHCPFFDLMKLFEVGGSPSNTRYLFLGDY~RGYFSlECVLYLUSLKINHPKTL . . . . . ..E........ V . ..-......-.................----..-....-.............

HUll Mur

FLLRGNHECRHLTDYFTFKPECRIKYSEPVYDACMETFDCLPL~LLNQaFLCVHGGMSPEITSLDDIRK . . . . . . . . . . . ..E.............. M . . . . ..H...........................

HUll Mur

LDRFTEPPAFGPVCDLLWSDPSEDYGNEKTLEHYTHNTVRGCSYFYSYPAVCEFLQNNNLLSlIRAHEAa . . ..s................ L . . . . S..................F............S...........

HUll Mur

DAGYRMYRKSPATGFPSLITIFSAPNYLDVYNNKAAVLKYENNMNIRQFNCSPHPYWLPNFMDVFTWSL 350 . .._..... N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 350

HU-II blur

PFVGEKVTEMLVNVLNICSDDELISDDEAEGSTTVRKEIIRNKIRAIGKMARVFSILR~ESESVLTLKGL . . . . . . . . . . . ..I......E.MNVT..-..A..G...V.K.............TV..E...N.......

420 419

HUll Mur

TPTGTLPLGVLSGGKPTlET----------AIRGFSLPHKIRSFEEARGLDRINERMPPRKDSIY--PGG . . . . . . . . . . . . . . . . . . ..AKPEAAEERE.....TIA.R . . . . . . . . . . . . . . . . . . . ..EAS.HHDA.

478 489

HUII Mur

PMKSVT-SAHSHAAHRSDPGKKAHS’ R.H.HSHPP.PQ.SR.T.H....L*

140 140 210 210

C-E....

280 280

502 513

Fiaure 2. Comparison of the deduced amino acid sequences of cDNAs corresponding to human and murine isoforms of the testis-specific catalytic subunit, PP28a3. The deduced sequence of the human cDNA ("Hum") (this paper) is aligned with that of murine cDNA ("Mur") reported in reference 10; spaces needed to optimize homology are indicated by hyphens. The cumulative number of residues for each form is indicated on the right. Positions of identity are indicated by periods.

isoforms

of the

variant exon

usage

testis, for

may give

rise

to

be selective study

Chromosomal

in our

mapainq

The genes have

been

that

their

for

assigned

of

if

the

gene for

the

testicular

genes,

to make its

sequence

under

the

chromosome linked

out

Southern

Because

all

three

three

genes. of

such

a

alternative subunit

mammalian

exon,

sequences

in genes

one can assume

for

the

Further,

alternative

there

appears in all

this

molecular

variant

of expression

of this

alternative

exon

blot

previously within

to

subunits,

4 and 10,

Because

isoforms analysis lack

of DNA from

268

(10).

PPZBal

gene

somatic

interest linkage

cell with

The majority (S.

suggesting

was of

The 122-bp

cross-hybridization

and PPZBd,

(13), it

showed any obvious

assignment. used

3 of the

PPZBal

respectively

physically.

of stringency exon

/X2&3: catalytic

human chromosomal

conditions

is contained

for

CaM-PrP

not

or other

genes,

"neural" is

was shown

catalytic

the

for

that

the

same spliced

maintenance

human qene

to human

carried

study

the

significance

the

the

these this

the

the

evidence

of

acquired

into

first suggests

laboratory.

expression

determine

enzyme

for

The functional

(8).

exhibit

divergence

pressure

genes. under

this

is the strongly

isoforms

in brain

subunit

its

this This

molecular

documented

gene for

prior

(8,14),

isoform. to

catalytic

an ancestral

three is

or testis,

CaM-PrP

splicing

subunit

a3,

as has been the

that to

catalytic

of the

Higuchi

to with

hybrids probe the

was

used other

of this

in two

probe

and R. L. K.,

Vol.

188,

No.

BIOCHEMICALANDBIOPHYSICALRESEARCH

1, 1992

t

COMMUNICATIONS

t

HIJ

Ha

Fiaure 3. Assignment of the testis-specific catalytic subunit gene to a human chromosome by Southern blot analysis of DNA from human-hamster hybrid cell lines. Nylon membranes containing DNA from human-hamster hybrid cell lines were obtained from Bios Corporation, New Haven, CT. Each lane containing 5 pg of Eco RIdigested DNA. Samples were applied in two regions of the agarose gel (11 samples at the top and 11 samples in the middle) and, after electrophoresis, the DNA was transferred to nylon membranes. Lanes containing human (Hu) and hamster (Ha) DNA were included in control lanes at either end of the blots (see outside lanes). The blot was hybridized to '"P-radiolabeled cDNA probe for the human PP2Bu3 (see Fig. 1). Hybridization in control lanes showed a 19 kb fragment that is specific for human (Hu) and a 9.4 kb fragment for the hamster (Ha) homologue. Hybrid cell lines which gave specific 19 kb fragments are indicated by arrows.

unpublished so far

data)

coincide

thus,

it

PrP

catalytic

hybrid

exactly

seems

corresponding cell

and the

likely subunit to

lines

the

exon/intron

with that genes. control

containing

those this

boundaries of al

(T.

of the

M. and R. L. K.,

area represents As shown in Fig. sample

of

chromosome

testis

human 8 (lanes

gene unpublished

determined data);

a conserved exon among the CaM3, a specific 19 kb fragment,

DNA (lane 6,7,9,and

1),

is

present

10) and is

in absent

four in

Vol.

188,

all

No.

BIOCHEMICAL

1, 1992

hybrids

lacking

this

kb EcoRl fragment to

chromosome, CaM-PrP

these

of

the

identical

to the

deduced

structures sequence

relatedness, at

this

about

the

subunit, (>99%)

gene

and human

sequences

11,12).

In

sequences the

are

similar

the

mammalian

rapidly,

to those

stringently in

presented

here

physiology

of

of

enzyme

of 30 amino

acids

some

Because

it

maturation,

may these

suggest events

from

form

present

at the

suggests aspect

best

species

of

its

this

in

the

shows

that are

of in

by a less

the

seen

terminus

divergence,

this that

identity

of

may be evolving function

testis

the

of murine

sequence

motility

differences

accommodated

the same

properties

isoform

the

gene

catalytic

carboxyl

tissue-specific that

the

identity

extreme

The reduced

was suggested

sequence

an ancestral

histidines,

that

of

data)

substitutions

despite several

was 80%

unpublished

study with

Although

(10).

and/or

that is

subunit

are essentially

forms,

Interestingly,

of the

similar

forms

the neural

and contains

subunits

isoform

their

neural

the

2).

that

germ-cell

however,

this

PP2Ba3:

of

and human forms

88% (Fig.

murine

testis

diverged two

only

in the

conserved.

the

than

charge

implies

Because

proteins of

lacking

8.

human PP28013 catalytic

(9).

these

contrast,

(r3 catalytic

and

a role

positive

the

a 9.4

hybridization

cells

and T. M. and R. L. K.,

either

differences.

has a net

from

for

to

Because

derived

gene

conserved

being

show hybridization DNA.

human and murine

of rodent

ORF, a region

striking

domain

that For

is much less

throughout displays

suggests

deduced

(7-9,

testis

(8,

PPZBa2 gene

the

same time.

the

of of the

human gene

to

also

on human chromosome

deduced

and 81% identical

the

COMMUNlCATlONS

RESEARCH

genomic

in samples that

resides

P/??Bal

lanes

hamster

seen

demonstrate

subunit

The overall

the

was never

data

catalytic

Comparison

from

BIOPHYSICAL

All

chromosome.

derived

19 kb fragment

the

AND

is

isoform sperm,

less

may play the

data

biochemistry

constrained

or

evolution

structure.

REFERENCES

:: 3. 4. :: 7. 8.

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BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

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