We identified a novel isoform transcript, MPP alpha-2, of the mouse Mg(2+)-dependent protein phosphatase (MPP) alpha gene. The amino acid sequence encoded by MPP alpha-2 differs from the previously known MPP alpha-1 sequence only at the carboxyl terminal region. Northern and in situ hybridization analysis revealed differential expression patterns of these two transcripts in the embryo and in the adult organism, suggesting an elaborate regulation of the MPP alpha gene

FIORENZA, Maria Teresa

HASUIKE S.

MIYAMOTO T.

WESTPHAL H.

ZHAO Y.

English

Archivio della ricerca- Università di Roma La Sapienza

Short sequence-paperMolecular cloning and expression analysis of MPPa-2, a novel mousetranscript detected in a differential screen of pituitary librariesToshinobu Miyamoto 1, Maria T. Fiorenza 1, Yangu Zhao, Shiga Hasuike, Heiner Westphal *Laboratory of Mammalian Genes and Development, National Institute of Child Health and Human Development, NIH, Bethesda, MD 20892, USAReceived 28 June 2001; received in revised form 19 February 2002; accepted 19 February 2002AbstractWe identified a novel isoform transcript, MPPa-2, of the mouse Mg2 + -dependent protein phosphatase (MPP) alpha gene. The amino acidsequence encoded by MPPa-2 differs from the previously known MPPa-1 sequence only at the carboxyl terminal region. Northern and insitu hybridization analysis revealed differential expression patterns of these two transcripts in the embryo and in the adult organism,suggesting an elaborate regulation of the MPPa gene.Published by Elsevier Science B.V.Keywords: Protein phosphatase; Differential screening; Lhx3; PituitaryThe LIM homeodomain proteins Lhx3 and Lhx4 areimportant regulators of pituitary development [1–6]. Thepituitary gland originates from two embryonic tissues. Theanterior and intermediate lobes are derived from Rathke’spouch arising from the oral ectoderm, the posterior lobefrom the neural ectoderm. The requirement for Lhx3 andLhx4 during early organogenesis was proven by genetargeting [3,4]. They are essential for normal developmentof Rathke’s pouch. In mutants that lack the function ofeither protein, there is an early arrest of pituitary develop-ment. In addition, both proteins are required for motorneuron development.In an effort to identify genes that mediate the control ofpituitary development exerted by Lhx3, we prepared cDNAlibraries from E12.5 wild-type and Lhx3 null mutant pitui-tary tissue. E12.5 corresponds to the onset of pituitarydifferentiation. Briefly, total RNA isolated from a singlewild-type and a single null mutant E12.5 pituitary wereconverted to cDNA and then amplified by LD-PCR. Shorterdouble-stranded cDNA fragments were generated by RsaIdigestion. cDNA fragments corresponding to mRNAs dif-ferentially expressed between the wild-type and the nullmutant pituitary were selected by way of a denaturation/hybridization procedure. Differentially expressed cDNAwas PCR-amplified using oligonucleotide primers specificfor adaptors linked to their ends. The subtracted mixture wascloned into the pGEM-T Easy cloning vector (Promega).cDNA inserts from randomly picked clones were individu-ally amplified. To identify clones that were differentiallyexpressed, cDNA blot arrays were prepared in duplicateusing the DNA amplified from each clone. The blots werehybridized with forward and reverse subtracted probes (referto user’s manual for details). Using the above procedure, weisolated an 800 bp clone. This clone was used to generate aprobe in order to screen an E12.5 whole embryo cDNAphage library (Stratagene). The screening resulted in theisolation of three clones that were sequenced in bothdirections. A 5V extension of one of these clones (ClonetechMarathon cDNA Amplification Kit) yielded cDNA with anopen reading frame corresponding to a protein of 326 aminoacids (Fig. 1). The first 317 codons correspond to MPPa-1,a member of a gene family known to encode Mg2 + -dependent protein phosphatases [7]. Thereafter, the sequen-ces of our cDNA diverge abruptly, revealing a C-terminusthat is distinct from that of MPPa-1. We use the termMPPa-2 to distinguish this putative protein from the onedescribed earlier. An amino acid sequence comparison ofthe two gene products is shown (Fig. 2).To elucidate the physiological role of MPPa-2, proteinphosphatase assay was carried out. To generate the fragmentof coding region of MPPa-2, PCR was performed with 5VRACE product as a template. The oligonucleotides used to0167-4781/02/$ - see front matter. Published by Elsevier Science B.V.PII: S0167 -4781 (02 )00293 -2* Corresponding author. Tel.: +1-301-496-1855; fax: +1-301-402-0543.E-mail address: hw@helix.nih.gov (H. Westphal).1 These authors contributed equally to the work.www.bba-direct.comBiochimica et Biophysica Acta 1577 (2002) 109–112amplify cDNAwere: 5V-CGGGATCCATGGGAGCATTTT-TAGACAAGCC-3V and 5V-GGGGCGGCCGCCTTATTTA-TTTTTGTTAATGATCCACCTTC-3V. The amplified prod-uct was digested with NotI/BamHI and cloned into pET21bvector (Novagen). The recombinant plasmid was transferredto the E. coli expression strain BL21 (DE3). MPPa-2 proteinwas purified and Western blot analysis was carried out withanti-His antibody as a primary antibody. A band with amolecular mass of 41 kDa was detected (data not shown).Protein phosphatase assay was performed using a Serine/Threonine Phosphatase Assay System (Promega) accordingto the manufacturer’s instructions. The reaction mixturecontained 50 mM imidazole (pH 7.2), 0.2 mM EGTA,0.02% h-mercaptoethanol, 0.1 mg/ml BSA, 0.1 mM phos-phopeptide and 5 mM MgCl2 or 1 mM EDTA. Base levelactivity ( < 0.1 pmol phosphate/min/Ag protein) was meas-ured in the control (vector alone). By contrast, the extractcontaining the vector with the MPPa-2 insert exhibitedsubstantial activity (3.16 pmol phosphate/min/Ag protein)in the presence of 5 mMMgCl2. This activity was reduced tobase levels ( < 0.1 pmol phosphate/min/Ag protein) in thepresence of 1 mM EDTA.Fig. 1. Nucleotide sequence and predicated amino acid sequence of the MPPa-2 gene. The nucleotide sequence has been deposited in GenBank under accessnumber AF369981.T. Miyamoto et al. / Biochimica et Biophysica Acta 1577 (2002) 109–112110In higher eukaryotes, four distinct MPP genes (PP2Ca,PP2Ch, PP2C x and Wip1) have been reported [8–12].Regulation of these genes seems complex as indicated bythe fact that at least four mRNA isoforms are derived fromthe PP2Ch gene, each with a distinct spatial and temporalpattern of expression. The encoded proteins differ from oneanother only in their C-terminal regions [13–15]. Recentstudies demonstrated that the alternative usage of twopromoters within the PP2Ch gene regulate the tissue-spe-cific expression pattern of PP2Ch mRNAs [16]. Recently,the human PP2Ca which inhibits the human stress-respon-sive p38 and Jun-N-terminal kinase (JNK) mitogen-acti-vated protein kinase (MARK) pathways was isolated [17].Our discovery of a second isoform of MPPa mRNAsuggests that the transcriptional regulation of this genemay be elaborate as well.Expression profiles of the MPPa-1 and MPPa-2 tran-scripts were established by section in situ hybridization ofthe E12.5 pituitary and surrounding tissues. Sagittal sec-tions (5 Am) of wax-embedded embryos were hybridized to33P-labeled riboprobes, according to published procedures[18]. RNA probes were generated using SP6 or T7 poly-merase (Ambion), using the manufacturer’s instructions.Exposure time for radioactive signal detection was 21 days.Riboprobes corresponded to distinct 3V regions of the twoMPPa transcripts. MPPa-2 was prominently expressed inthe E12.5 wild-type pituitary and the adjacent floor of thediencephalon but not in the Lhx3 null mutant pituitary (Fig.3A, B), thereby validating the approach that led to theisolation of this transcript. By contrast, MPPa-1 transcriptswere detected in the E12.5 pituitaries of both wild-type andLhx3 null mutant embryos (Fig. 3C, D). This implies thatthe transcriptional regulation of the MPPa gene is part ofthe pituitary differentiation program controlled by Lhx3.A mouse multiple tissue Northern blot (Clontech) washybridized with 242 and 800 bp probes generated by PCRFig. 3. E12.5 pituitary (arrows) expression patterns of MPPa-2 (A, B) and MPPa-1 (C, D) in wild-type (A, C) and Lhx3 null mutant embryos (B, D),respectively.Fig. 2. Comparison of the deduced amino acid sequences encoded by MPPa-1 and MPPa-2 transcripts, respectively. Asterisks show the positions of stopcodons. Bold face indicates sequence divergence.T. Miyamoto et al. / Biochimica et Biophysica Acta 1577 (2002) 109–112 111from 3V sequences specific for the MPPa-1 and the MPPa-2transcript, respectively. Differential regulation of MPPatranscription is clearly not restricted to the developingpituitary. A Northern analysis of select adult tissues revealedthe presence of MPPa-1 mRNA in heart, liver and testis(Fig. 4A), and that of MPPa-2 mRNA in heart, brain andliver (Fig. 4B).In conclusion, our results show a novel isoform ofPP2Ca. PP2Ca-2 is expressed during wild-type pituitarydevelopment, but this expression is not detected in the Lhx3mutant. This differential expression pattern is not seen withPP2Ca-1 indicating distinct transcriptional control mecha-nisms for these isoforms.AcknowledgementsT.M. was supported by a fellowship of the JapaneseSociety for the Promotion of Science.References[1] M. Taira, W.P. Hayes, H. Otani, I.B. 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Molecular cloning and expression analysis of MPPa-2, a novel mouse transcript detected in a differential screen of pituitary libraries

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Molecular cloning and expression analysis of MPPa-2, a novel mouse transcript detected in a differential screen of pituitary libraries

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