AbstractA 1.0 kilobase cDNA coding for the complete amino acid sequence of a putative protein phosphatase (314 amino acid residues, molecular mass 36 kDa) has been isolated from a Drosophila head cDNA library. The cDNA hybridises to a single site on the right arm of the second chromosome at cytological position 55A1–3. The deduced sequence of the protein, designated protein phosphatase-Y, is homologous to the catalytic subunits of Drosophila and rabbit protein phosphatase- 1α (64 and 59% identity, respectively) and rabbit protein phosphatase-2A (39% identity). These and other comparisons demonstrate that this novel enzyme is not the Drosophila counterpart of mammalian protein phosphatases 1, 2A, 2B, 2C or X

Axton, J.Myles

Cohen, Patricia T.W.

Dombrádi, Viktor

Glover, David M.

Elsevier - Publisher Connector 

Volume 247, number 2, 391-395 FEB 07 105 April 1989 
Molecular cloning and chromosomal ocalization of a novel 
Drosophila protein phosphatase 
Viktor DombrBdi, J. Myles Axton *, David M. Glover* and Patricia T.W. Cohen 
MRC Protein Phosphorylation Group, Department of Biochemistry, Medical Sciences Institute, University of Dundee. 
Dundee DDI 4HN, Scotland and *CRC Eukaryotic Molecular Genetics Research Group, Department of Biochemistry, 
Imperial College of Science, Technology and Medicine, London S W7 ZAZ, England 
Received 15 March 1989 
A 1.0 kilobase cDNA coding for the complete amino acid sequence of a putative protein phosphatase (314 amino acid 
residues, molecular mass 36 kDa) has been isolated from a Drosophila head cDNA library. The cDNA hybridises to 
a single site on the right arm of the second chromosome at cytological position 55Al-3. The deduced sequence of the 
protein, designated protein phosphatase-Y, is homologous to the catalytic subunits of Drosophila and rabbit protein 
phosphatase-la: (64 and 59% identity, respectively) and rabbit protein phosphatase-2A (39% identity). These and other 
comparisons demonstrate that this novel enzyme is not the Drosophila counterpart of mammalian protein phosphatases 
1,2A, 2B, 2C or X. 
Protein phosphatase; cDNA cloning; Nucleotide sequence; Amino acid sequence; (Drosophila melanogaster) 
1. INTRODUCTION 
Reversible protein phosphorylation is a post- 
synthetic modification that is used widely in euka- 
ryotic cells to transduce extracellular stimuli [l]. 
The degree of phosphorylation of a specific protein 
depends on protein phosphatases as well as protein 
kinases. Four serinejthreonine-specific protein 
phosphatases have been characterised by enzymatic 
and physiochemical methods and termed PP-1, 
PP-2A, PP-2B and PP-2C [I]. Molecular cloning 
has been used successfully to reveal the primary 
structures of the catalytic subunits of these en- 
zymes. Two forms of PP-1 were found in rabbit 
and Drosophila [2-61. PP-2A has been sequenced 
from mammalian sources [7-131 and two isozymes 
Correspondence address: P.T.W. Cohen, Department of 
Biochemistry, Medical Sciences Institute, University of Dundee, 
Dundee DDl 4HN, Scotland 
Abbreviations: PP, protein phosphatase; kb, kilobase; SSC, 
0.15 M NaCl, 0.015 M sodium citrate, pH 7.0 
The nucleotide sequence presented here has been submitted to 
the EMBL/GenBank database under the accession o. YO7510 
have been detected in rabbit [7,8], porcine [I l] and 
human tissues [131. The partial sequence of murine 
PP-2B [14] and a novel rabbit enzyme termed PP- 
X [9] have also been determined. The latter is more 
similar to PP-2A than to any other phosphatase. 
Here, we report on the identification by recombi- 
nant DNA techniques of another novel phospha- 
tase in Drosophila, which has greater similarity 
with PP-1 than PP-2A. 
2. EXPERIMENTAL 
2.1. Screening 
A Drosophila head cDNA library, constructed in hgtl0, was 
screened with a 0.76 kb SmaI/NaeI rabbit PP-1 cDNA fragment 
[3] coding for amino acids 43-298, and with a 51-base synthetic 
oligonucleotide [2] coding for amino acids 152-168 of rabbit 
skeletal muscle PP-la. The cDNA probe was labelled with 
[y”*P]dCTP using the random oligonucleotide priming method 
[15] to a specific activity of -2 x 10’ dpm//g and further 
purified by spun column chromatography [16]. The labelled 
cDNA was hybridised at a concentration of 0.5 ng/ml to recom- 
binant DNA on nitrocelluiose filters as described [9]. The filters 
were washed with 0.2 x SSC containing 0.1 % SDS at 65°C. 
The oligonucleotide probe was labelled with [y3*P]ATP by Tq 
polynucleotide kinase [16] to a specific activity of -4 < lo6 
dpm/pmol. Hybridization was carried out with 0.8 pmol/ml 
Published by Elsevier Science Publishers B. K (Biomedical Division) 
00145793/89/$3.50 0 1989 Federation of European Biochemical Societies 391 
Volume 241, number 2 FEBS LETTERS April 1989 
labelled oligonucleotide according to [17]. The filters were 
0 0 washed in 6 x SSC at 5O’C. 
o -” 
2.2. Subcloning and sequencing 
The inserts of two clones selected by hybridisation to the rab- 
0 200 400 600 800 1053 
Fig. 1. Strategy used to sequence the cDNA coding for 
Drosophila protein phosphatase-Y. The scale indicates the 
nucleotide position in base pairs starting from the 5 ‘-end. The 
arrows show the direction and length of the sequences obtained 
with Bluescript primers (0) and specific oligonucleotide 
primers (0). 
bit cDNA, but which did not hybridise to the oligonucleotide 
probe, were subcloned into Bluescript pKS+ vector (Stratagene 
Cloning Systems, San Diego, CA). DNA sequencing was per- 
formed on the denatured ouble-stranded plasmid preparations 
by the dideoxy chain-termination method [18] using synthetic 
oligonucleotide primers (fig.l), modified T7 DNA polymerase 
(US Biochemical Corp., Cleveland, OH), [(u”S]dATP and buf- 
fer gradient gel electrophoretic separation [19]. 75% of the se- 
quence was also determined with 7-deaza-2 ’ -dGTP in order to 
resolve compressions in the G-C rich regions (201. 
ATAAT 
ATGGCCGTCTTAACTACTCATGAAATAGAT TGCATTATAMGGAGCTCACATCTCTGAAT~ 
MAVLTTBEID CIIKELTSLN 
GGAAGTGAGTGCWATTIGAGGAACTA ATCGACAGACTCATTCMCACACTCGTGAA 
GSECTLKEEL IERLIQQTRE 
GTGAT~TGGCAACCGGCTGCTGGAA CTTCAGGCTCCGGTCAATGCGGCGGCGAT 
v I KW Q PM L L E LQAPVNI C G D 
ATTCATCCCCAGTTTACAGATCTCTTGAGG ATTTTCAAGGUTGCGGCTTTCULCCCCAAA 
I H GQ F T D L L R IFKACGFPPK 
GCCAACTATTTATTTCTCGGTWLCTATGTG GACCGAGGCiMGCAATCGCTGGAAACGATT 
ANYLFLGDYV DRGKQSLETI 
TGTTTGCTATTTGCTTACAAAGTTllAATAT CCGCTAAATTTCTTTTTGCTTCGCGGCAAT 
CLLFAYKVKY PLNFFLLRGN 
365 
120 
CACGAGTGCGCCAGTATAAAAATTTAC 
li E CA S IN X I Y 
ACTGTCCGTTTGTGGCACTTCACGGAT 
TVRLWBNFTD 
GGATTTTACGACGAGATCAAACGTAGACAC 
GFYDEIKRRH 
425 
140 
TGCTTCAACTGGCTTCCGGTGGCCGCGTTG 485 
CFNWL.PVAAL 160 
GTGGGCGAGCGCATCTTCTGCTGCCACGGA 
V G E R I F C C I1 G 
CAGATCAATCATATTCAGCGACCCACTGAT 
QINHIQRPTD 
GGACTGAGTCCATCGCTCCGGAATTTGCAA 
GLSPSLRNLQ 
545 
ia0 
ATTCCGGATGAGGGTATTATGTGCGATCTC 605 
IFDEGIMCDL 200 
CTTTGGGCGGATCTAAATCACACCACCAAA GGCTGGGGTCACAACGATCGCGGTGTGAGC 665 
LWADLNBTTK GWGSNDRGVS 220 
TTCACCTTCGATMGGTCATAGTTCGGGAT TTTCTTAAAGCCTTCGACTTGCAWTTATG 725 
FTFDKVIVRD FLKAFDLQLM 240 
GTTCGCGCCCATGAGGTTGTGGAGGATGGA TACGAGTTCTTTGCCAACCGACAGCTGGTC 785 
VRAHEVVEDG YEFFANRQLV 260 
ACCGTATTCTCGGCCCCCAACTATTGCGGT 
TVFSAPNYCG 
ATGATGAACAATGCCGGCGGAGTGATGAGT 
MMNNAGGVMS 
GTTAGCACAGACTTGATCTGCTCCTTCGTC 
V S T D L I C SF V 
ATTGCGACTGATGCCAACCTGCCGACT 
IATDANQHPT 
ATTATTCTACCGTGTCACAAATACAAAATG 
IILPCSKYKM 
MCGAAGAGGAG 
NE E E 
a45 
280 
905 
300 
947 
314 
TGATTTTGTTTTTCATACATGTATATATCA CTTCGTGCAATTAAATTCGTACAATAACAA 1007 
TTCTTTTTATGTAGCTGATATATC 1031 
5 
65 
20 
125 
40 
185 
60 
245 
a0 
305 
100 
Fig.2. Nucleotide sequence and predicted amino acid sequence of Drosophila protein phosphatase-Y. 
392 
Volume 247, number 2 FEBS LETTERS April 1989 
Table 1 
Comparison of the amino acid sequence of Drosophila PP-Y to other 
known protein phosphatase sequences 
Enzyme Number of 
amino acids 
compared 
Identity Homology 
(olo) (Q) 
References 
Drosophila 
PP-lo 
PP-l/3 
300 61 IO 151 
129 64 74 161 
Rabbit 
PP-la 
PP-2Aa 
PP-2/@ 
PP-x 
314 59 68 ]31 
309 39 52 171 
309 39 52 181 
203 38 51 191 
Murine 
PP-2B 111 32 47 1141 
PP-1 
PP-i 
PP-2A 
PP-1 
PP-Y 
PP-2A 
PP-1 
PP-Y 
PP-PA 
PP-1 
PP-Y 
PP-PA 
PP-1 
PP-Y 
PP-2A 
PP-1 
PP-Y 
PP-PA 
PP-1 
PP-Y 
PP-2A 
PP-1 
PP-Y 
PP-PA 
PP-1 
l *GWGENDRGVSF*FG**VV*KFL**H Li""l'[;DKD ncz fl a 
IHCDLLWADLNHTTKGWGHNDRGVSFTFDKVIVRDFLKAF 
PMCDLLWSDPD-DRGGWGISPRGAGYTFGQDI*ETFNN*N 
40 
36 
33 
80 
76 
73 
239 
235 
232 
279 
275 
272 
Fig.3. Comparison of animal protein phosphatase sequences. Amino acids that are variable among different species and isoenzymes are 
represented by asterisks. Residues identical in PP-Y and at least one of the compared protein phosphatases are boxed. The sequences 
were obtained from the following sources: PP-1 rabbit muscle [2-4, 261, Drosophila head [5,6]; PP-Y Drosophila head (this paper); 
PP-ZA, rabbit muscle [7,8] and liver [9], bovine adrenal [lo], porcine kidney [ll], rat liver [12], human liver [13]. 
393 
Volume 247, number 2 FEBS LETTERS April 1989 
54 1 55 1 56 ) 57 
1Opm 
Fig.4. Localization of Drosophila protein phosphatase-Y by in 
situ hybridisation to salivary gland polytene chromosome 2. The 
arrow indicates the site of hybridisation at S5A l-3. 
2.3. In situ hybridisation 
The entire PP-Y cDNA insert (fig.2) was labelled with 
[‘H]dCTP and [3H]TTP by the random oligonucleotide priming 
technique [15] to 2 x 10’ dpm//g specific activity. The probe 
was hybridised to polytene chromosome preparations obtained 
from the salivary gland of female Canton S strain D. melano- 
gaster larvae as described [S] . 
3. RESULTS 
Out of 216 000 plaque forming units of a Droso- 
phila head cDNA library we found 26 clones that 
hybridised to a 0.76 kb rabbit PP-1 cDNA probe 
but were not detected by a synthetic oligonu- 
cleotide coding for a rabbit PP-1 tryptic peptide. 
From these, two clones containing 1.0 kb inserts 
were purified and sequenced (fig.1). These clones 
were identical and contained the full-length coding 
region of a protein of 3 14 amino acids. The 5 ’ -non- 
coding region is extremely short and the 3 ‘-non- 
coding region has 3 additional potential stop 
codons starting at positions 989, 1002 and 1023, 
one of which is in-frame with the stop codon at 948 
(fig.2). The predicted molecular mass of the encod- 
ed protein is 36 026 Da assuming that there are no 
posttranslational modifications. The deduced 
amino acid sequence is distinct from any known PP 
sequence (table l), but nevertheless contains most 
of the polypeptide stretches that are highly con- 
served among the members of the PP-l/PP-2A 
family (fig.3). This clone hybridises in situ to 
polytene chromosomes at a single locus on the right 
arm of the second chromosome at cytological posi- 
tion 55Al-3 (fig.4) on the standard maps 1211. 
4. DISCUSSION 
We predict that the protein encoded by the clone 
described here (fig.2) and designated PP-Y is a new 
member of the PP family. Although isolated with 
the aid of a PP-1 cDNA probe, it probably does 
not represent a closely related isoenzyme of PP-1 
for the following reasons. (i) The amino acid se- 
quence of PP-Y is distinct from PP-la (table 1, 
fig.3) and a recently sequenced fragment of 
Drosophila PP-lfl, a new PP-1 isoenzyme (table 1). 
On the other hand, PP-la and PP-lp are very 
closely related and both are highly conserved (92% 
identity for PP-lcu) from mammals to Drosophila 
[6]. (ii) The nucleotide sequence of the PP-Y 
coding region shows only 63% identity to that of 
Drosophila PP-la [5]. (iii) The Drosophila PP-1 
genes were located on the right arm of chromosome 
3 and on the X-chromosome by in situ hybridisa- 
tion [5], while the PP-Y gene mapped on the se- 
cond chromosome. (iv) All published PP-1 
nucleotide sequences are G-C-rich especially at the 
third positions of the codons [2-51, in contrast to 
PP-Y which has an average G-C content (47% in 
the coding region and 55% in the third positions of 
the codons). 
It is unlikely that PP-Y is a type-2 PP, since it 
has only about 50% amino acid homology to 
PP-2A and PP-2B (table l), nor did we find any 
homology with the sequenced peptides of PP-2C 
(comprising -65% of the total sequence) [22]. 
Drosophila PP-2A has been sequenced and local- 
ized at a different cytological position (Orgad, S., 
Dudai, Y. and Cohen, P.T.W., unpublished 
result), and the molecular mass of the PP-2B 
catalytic subunit [23] is much higher than that 
predicted for PP-Y. Furthermore, the amino acid 
sequence of PP-Y is different from that of PP-X, a 
novel hepatic PP identified by molecular cloning 
(table 1). These considerations indicate that PP-Y 
is a related but quite distinct protein. 
Since PP-Y was found in a head cDNA library 
and has the highest homology to PP-1 catalytic 
subunits it is possible that PP-Y is a neuronal form 
of PP-1. Alternatively, it could be an enzyme 
related to smooth muscle phosphatase IV which 
has a substrate specificity similar to PP-1, but is 
unaffected by inhibitor-2, a protein which inhibits 
PP-1 specifically [24]. A further possibility is that it 
may be highly specific for a substrate that has yet 
to be identified, like the protein phosphatase 
reported to be specific for ribosomal protein S6 
[25]. Expression of the clone and subsequent 
analysis will be necessary to identify its enzymatic 
properties and physiological role(s). 
394 
Volume 247, number 2 FEBS LETTERS April 1989 
Acknowledgements: Thanks are due to Dr D. Kalderon and 
Professor GM. Rubin (University of California, Berkeley, CA) 
for the Drosophila cDNA library, and to Mr A.I.H. Murchie 
(University of Dundee, Dundee, Scotland) for synthesis of 
oligonucleotides. This work was supported by a grant from the 
Wellcome Trust and Group Support from the Medical Research 
Council, London (to P.T.W.C.) and a grant from the Cancer 
Research Campaign (to D.M.G.) V.D. was supported by a 
Long-Term EMBO Fellowship and is on leave from the Institute 
of Medical Chemistry, University School of Medicine, Debre- 
ten, Hungary, J.M.A. holds a Science and Engineering 
Research Council Studentship. 
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395 


Molecular cloning and chromosomal localization of a novel Drosophila protein phosphatase 

A 1.0 kilobase cDNA coding for the complete amino acid sequence of a putative protein phosphatase (314 amino acid residues, molecular mass 36 kDa) has been isolated from a Drosophila head cDNA library. The cDNA hybridises to a single site on the right arm of the second chromosome at cytological position 55A1–3. The deduced sequence of the protein, designated protein phosphatase‐Y, is homologous to the catalytic subunits of Drosophila and rabbit protein phosphatase‐ 1α (64 and 59% identity, respectively) and rabbit protein phosphatase‐2A (39% identity). These and other comparisons demonstrate that this novel enzyme is not the Drosophila counterpart of mammalian protein phosphatases 1, 2A, 2B, 2C or X

Axton, J. Myles

Cohen, Patricia T. W.

Caltech Authors - Main

Molecular cloning and chromosomal localization of a novel Drosophila protein phosphatase

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Molecular cloning and chromosomal localization of a novel Drosophila protein phosphatase

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