Proc. Natl. Acad. Sci. USA Vol. 90, pp. 4882-4886, June 1993 Biochemistry Identification and cloning of a kinase-encoding mouse gene, Plk, related to the polo gene ofDrosophila (serine-threonine kinase/polo-like kinase) FIONA J. CLAY*, STEPHEN J. MCEWEN*, IVAN BERTONCELLOt, ANDREW F. WILKS*, AND ASHLEY R. DUNN*t *Melbourne Tumour Biology Branch, Ludwig Institute for Cancer Research, PO Royal Melbourne Hospital, Victoria 3050, Australia; and tPeter MacCallum Cancer Institute, 481 Little Lonsdale Street, Melbourne, Victoria 3001, Australia Communicated by G. J. V. Nossal, January 14, 1993 (received for review November 23, 1992)

ABSTRACT We have determined the nucleotide sequence to emerge from this study encodes a protein kinase that of a cDNA encoding a protein kinase that is closely related to shares extensive homology with the enzyme encoded by the the enzyme encoded by the Drosophila melanogaster mutant Drosophila melanogaster mutant polo (11).§ polo and that we have designated Plk (polo-like kinase). Plk is also related to the products of the cell MATERIALS AND METHODS cycle gene MSD2 (CDC5) and the recently described early growth response gene Snk. Together, Plk, polo, Snk, and DNA Amplification. Poly(A)+ mRNA prepared from puri- MSD2 define a subfamily of serine/threonine protein kinases. fied primitive hemopoietic cells isolated from the bone mar- Plk is expressed at high levels in a number offetal and newborn rows of C57BL/6 mice (12-14) was used to generate cDNA, in the corresponding adult using a cDNA Synthesis System Plus kit (Amersham). PCR mouse tissues but is not expressed was performed with the degenerate oligonucleotide primers organs. With the exception of adult hemopoietic tissues, the PTK1 and PTKll (10). PCR and the cloning ofPCR products only adult tissues in which we could detect Plk expression were were performed as described (10). ovaries and testes. Taken together, the patterns ofPlk expres- Molecular Cloning and Sequence Analysis. A mouse sion suggest an association with proliferating cells. Since polo BALB/c bone marrow cDNA library in Agtll (Clontech) was is required for mitosis in Drosophila it is possible that Pik is screened with the radiolabeled PCR-generated product (see involved in some aspect of cell cycle regulation in mammalian Results), using standard hybridization conditions at high cells. stringency (15). The insert of clone BM-1 was used to screen a murine ICR-CD1 anemic spleen cDNA library in Agtll The coordinated control of cell growth and differentiation in (Clontech). We isolated two overlapping cDNA clones, S15 is achieved, in part, through the activation of and S13, which encompassed the catalytic domain and the 3' intracellular biochemical networks in response to external , respectively. To isolate a cDNA corre- stimuli. Protein kinases are known to represent integral sponding to the 5' end ofPlk, a mouse neuroepithelial cDNA components of many signal transduction pathways; not only library (strain DBA) in Agtl0 (16) was screened with a do they serve to phosphorylate their normal physiological radiolabeled Bgl II fragment (nt 675-771) recovered from the their enzymatic activity is, in turn, regulated 5' end of S15. One clone, NE4, contained a 1.5-kb insert. substrates, Inspection of the nucleotide sequences of cDNA clones S15 through interaction with other protein kinases and phos- (strain ICR-CD1) and NE4 (strain DBA) in the regions of phatases (1-3). Homologues of a large number of protein overlap revealed a number of sequence polymorphisms, kinases have been identified from a range of eukaryotic some of which were conservative, while others resulted in species including mammals, budding and fission yeast, and amino acid substitutions. Since we were unable to isolate Drosophila, suggesting that at least some of the processes further cDNAs that encompassed the N terminus of Plk we that govern cell growth and differentiation in this diverse performed PCR on liver DNA from random-bred ICR-CD1 range of organisms are achieved by using similar principles mice, using 5' sense (5'-CCCAAAGCTTGTGTCTGAC-3') (2-5). and 3' antisense (5'-CTGCGAACACCTCTTTTG-3') oligo- In general, eukaryotic protein kinases are classified on the nucleotides based on the nucleotide sequence of NE4. Nu- basis of substrate specificity (4). Recently a number of cleotide sequence analysis ofboth strands was carried out by protein kinases have been identified which have the dual using the dideoxynucleotide chain-termination procedure capacity to phosphorylate serine/threonine and tyrosine res- (17) either manually or with an Applied Biosystems auto- idues (3, 6, 7). Inspection ofthe primary sequences ofprotein mated sequencer. The DNA sequence and deduced protein kinases reveals the presence ofparticular sets ofresidues that sequence were compared with the GenBank data bases by are invariant features of molecules with the capacity to using the FASTA programs (18). phosphorylate specific substrates (8). Interspersed among Northern (RNA) Analysis. Total cellular RNA was isolated the motifs that signify serine/threonine or tyrosine specificity as described (19), fractionated on 1% agarose/formaldehyde are shorter stretches of amino acids that are characteristic of gels, and transferred to GeneScreenPlus membrane (DuPont) subgroups of protein kinases and which presumably contrib- according to manufacturer's protocols. ute to the substrate specificity and/or the manner in which individual protein kinases are regulated (4, 9). We have employed a PCR-based strategy (10) to isolate RESULTS sequences corresponding to protein kinases expressed in Isolation of Clones Encoding Plk. To isolate protein kinases primitive murine hemopoietic progenitor cells. One sequence that may play a role in the regulation of hemopoiesis, we

The publication costs of this article were defrayed in part by page charge iTo whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" §The sequence reported in this paper has been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. L06144). 4882 Downloaded by guest on September 28, 2021 Biochemistry: Clay et al. Proc. Nati. Acad. Sci. USA 90 (1993) 4883

isolated a highly enriched population of primitive hemopoi- deduced amino acid sequence of P1k are shown in Fig. 1 etic progenitor cells from mouse bone marrow (12-14). Se- Lower. quences corresponding to putative protein kinases were Sequence of Plk. The nucleotide sequence of Plk contains amplified by PCR using the degenerate oligonucleotide prim- a single open extending over 1809 nt -and ers PT7K1 and PTK2 on cDNA reverse transcribed from encoding a p'rotein of 603 aa (Mr, 68,409). The nucleotides mRNA isolated from these cells (10). PTK1 and PTK2 flanking those encoding the residue at position correspond to the conserved subdomains Vl and IX in the 470-472 conform to the Kozak consensus sequence for the catalytic domain of protein kinases as defined by Hanks et al. initiation of protein (20). Furthermore, an in- (4). The products of this reaction were subcloned in frame termination codon is located 105 nt upstream of the M13mp18 and the nucleotide sequences of randomly chosen proposed AUG. clones were determined. A number of previously described Relationship ofPlk to Other Protein Kinases. Comparison of protein tyrosine kinases were identified, as well as one novel the deduced amino acid sequence of P1k with other published sequence that on the basis of amino acid sequence encoded sequences revealed a close relationship between P1k and a protein with features of a serine/threonine protein kinase. three serine/threonine protein kinases. The catalytic domain A number of overlapping cDNA clones (SiS and S13) as well of P1k is most closely related to the polo gene product (65% as a genomic fragment (PCR-A) (Fig. 1 Upper) corresponding identity), a protein kinase required for mitosis in Drosophila to the novel sequence were isolated according to the strategy (11,21) (Fig. 2). Plk and polo encode of a similar size outlined in Materials and Methods. The composite nucleo- and have a similar overall structure, with their catalytic tide sequence [S15, S13, and PCR-A (Fig. 1 Upper)] and domains being located in the N-terminal region. P1k is also

ATG TAG WV Kinase Domain 0i

BGLII TAG S15 I II IWlw

S13 v I HINDuI ATG BGLII I I V i NE4 LU HINDHI ATG BGLII 100 bp PCR-A IV II

1 AGCTGTOAGTCTGAAAGTTOCCTOGCTGAOCTTGAAO ATCTAGACATOOOTOTOGOCTOCAACAAOOOOCCTCATCTCATCACTCATTGGTCTTATA 101 TOMrrATTTTTACCAGCCTGTTCTOGAOTOTO3OCAOOOOCATOOCCTOGCTTCCCATCAGGAGAAATCAGCAGAAGTOGACAGOGOTACCTOTATC 201 CACAOOTGCCAGCTOOCCTGTOOGTTAGOTACAAGOGCOTGOOCACAOCTCCTTCCTCAGOCTACCTC173CTGAG&CCTCAGCCCTAOGCTATACCTTOA 301 AGCAGCCJGAGOAGAOTCTOOGCATAAAGGCCCAGCAAAATTATOTTOTCCTACTOTTOTGCTCTGAGCCCCCATTTCTOvCCATTTrOCTOCATATCAAA

A V A K A 0 K L A 11 401 0c2AAAAGAAACCATCGCACGAOGACCCTCTCCACCGACTCAGCCAGGTTCGTOOAOCAACTTCQOCATCATOAALTOCATGGCCAAAOCTOGAAAAOCTO

R AP TDLOGKOOGV LOGD A AP SA PV A AP L AKE I L EV L 44 501 aTCGAGCACCAACCGACCTCOGGAAAOGTOGOGTCCTOOGAG&TOGCAOCTCCCAOTOCCCCCAGTOOCTOCCCCCACTOGCGAAAAGAATTrCTOGAOOTCCT

V D P R S R RQ I R R F L K F A K C F E I S D A D T K E 77 601 AGTOGACCCACOCAGCCOGCOOCAGTATATACGGOOCCGCTTTCTOOGTAAAOOAOOCTTCGCCAAATOGCTTCGAGATCTCAGACOCAGACACAAAAGAG V F A G K I V P K S L L L K P H Q K E K H S H E I S I H R S L A H Q 111 FIG. 1. (Upper) Schematic 701 TOTCGCAOOCAAGATCGTGCCTAAGTCTTTGCTOCTCAAOCCCCACCAGAAGGAGAAGATOTCTATOGAGATCTCAATTCACCGCAOCCTAGCACACC representation of the predicted structure of the P1k H VV G FH D FFE D SD F VFV V LE LCRR R SLL E LHKR 143 composite 801 AACACGTCGTAGOCTTCCATOAC rOAOGACAOCOAC?TATTGTATTOAOTTTTOGAGCCTCTTCGCAOOAGOTCCCTCCTOGAOCTOCACAAGAG mRNA flanked by the 5' and 3'

RK A LT EPE ARY Y LRQ IV L GCQ Y LHRHNQ V IH RD L 176 untranslated regions. The three 901 GAGAGA7ACAC7AOCGTCACGGCGTGCTGCGCGACICCOATAGCTCCGGC7 overlapping cDNA fragments and K L 0 N L F L N E D L E V K I G D F G L A T K V E Y E 0 E R K K T L 210 the genomic PCR clone used in 1001 this study are shown below. The COGT PHN Y I A P E V L S K K O H S F E V D VW S I G C I H Y T L 243 coding region is indicated by the 1101 TOGTOTOOCACTCCTAACTACATAGCTCCTGAGGTGCTOAGCAAGAAGGGAACAGTTTAO? G,-AO.TGTOGTOGTCCATTGOGOTCATCATGTATACCTT box. The kinase (catalytic) domain L V G K P P F E T S C L K E T Y L R I K K H E Y S I P K H I H P V 276 is indicated by the shaded region 1201 within the box. The HindlIl and A A S L I Q K H L Q T D P TA R P T I H E L L H 0 E F F T S G Y I P 310 II sites used in the of 1301 GCCGCCTCCCTCATCCAGAAGATOCTTCAGACAGACCCCACTOGCCCGCCCCACCATTCACGAGTTGCTCAATOGACOAGTTCTTCACTTCTOOCTACATCC Bgl cloning the PCR fragment are indicated. A R L P I T C L T I P P RF S I A P 5 5 LD P 5 5S R K P L K V L H 343 1401 CCGCCCGTCTCCCTATTACCTGCCTCACCATCCCACCAAGG?TTTCAATCGCTCCCAGCAGCCTOGACCCCAOCAGCAOGAAACCTCTCAAAGTCCTCAA (Lower) Nucleotide sequence of K 0 V E H PL P D R P R E K EE P V V R E T H E A I EC H L S D L 376 murine Plk and deduced amino 1501 TAAAGGTOTGGAGAACCCCCTOGCCTGACCGTCCCCOOOAGAAAGAOGAACCGGTOGTCCGGGAOACAAATOAGGCCAT7rAGTOCCACCTTAGTOACTTOG acid sequence. Nucleotides are L0Q L T S V H A S K P 5 E R 0 L V R Q E E A E D P A C I P I F WV 410 numbered to the left of the se- 1601 CTACAGCAGCTOACCAGTOTCAACGCCTCCAAGCCCCTCGGA A0COCGG CTOGTOCOOCAAAGGAOO,AGaCCToACCA¶GCATCCCATCTTCToG quence; amino acids are num-

SK WV D YS D KYOGLOGY Q LC DHNSVOGVL FHND ST RL I L 443 bered to the right of the protein 1701 TCAGCAAGTOOOTOOGACTATTCOGACAAGTATGGCCTI'IOOTATCAGCTGTOGTGACAACAGTGOToOTOCI'I'ITTAATOACTCAACACGCCTGATTrCT sequence. The positions of the oil-

YHNDOGD S L QY I ERDOGT E SY LTV S SH PHNS LHNKK I T 476 gonucleotide primers used in the 1801 CTACAATOACGOGOACAGCCTOCAGTACATAGAGCGTOATOOGCACGGAGTCCTATCTCACTGTGAGCTCCCATCCCAMTTCCTrOATOAAGAAGATCACT original PCR screening are indi- H H H 0 H 0 L L Y F R Y S E H LL K A R I T P R E D E L A R L P Y L 510 cated (10). Bent arrows define the 1901 CTCCTCAACTATTCCGCAATTACATGAGTGAG.CACCTO.CTGAAGGCAGGACOCAACATCACACCCCGGOAAGGCOACGAGCTGGCCCOGCTOGCCCTACC catalytic domain. The 5' upstream R T W F R T RS A I I L H S H 0 H F L T V Q I F Q DH T K L I L C 543 in-frame termination codon and 2001 TACGAACGTOGTTCCGCACACGCAGCGCCATCATCCTOCACCTCAGCAACOOCACCGTOGCAGATTAACTTCITCCAOOGACCACACCAAACTTATCCTGTG the consensus polyadenylylation P LHMA AVT YIHNE KR D FQT YRL SL LE EYOGC CK EL A 576 are underlined. The Hind- 2101 CCCCCT¶ATGGCAGCOGTGACCTACATCAACOAaAAGAOGACTTCCAAACGTACCGCCTAOCCCCTCCo9AOGAGTATOOcTCTGC7AAGGAGCTOOCC signals III (partial recognition sequence) S R LRY AR TMV DK LLS SR SA SHNRL K A S 603 2201 AGCCOCCTCCGCTACGCCCGCACCATOOTAGACAAGCTOGCIOOCTCGCGCT'CCGCCAGCAACCOCCTCAAOOCCTCCTAOOTCTCTCTCCTTCOGACTO and Bgl II sites shown in Upper 2301 GTOCCCCTTCACT~CTAGCTACT0rAOCTQCACTOGTA0OCTCCTOOOOOCTGCT0TIAGTroCCCCT0OCCCTOOOOCT0OOCAOOAOCA~OOCCCCC 2401 ITIAOOGGG T0OTTAAGTTA?TTTTTACATGTCTr.OGTGTOOTTTACATCTr.CTTCCCTOCCCTCGCTCAGCCCACTOTATGAAITTTATAA are located at nucleotides 1-5 and 2501 AIrTATTCTATTAAATOGACTOCT TTTCCTCTTAGCTTATATATATTAAAATGG 2555 675-681, respectively. Downloaded by guest on September 28, 2021 4884 Biochemistry: Clay et al. Proc. Natl. Acad. Sci. USA 90 (1993)

GG V A K E Plk (53) polo (25) MSD2 (82) Snk (79) Subdomains

H HRDLK N K DFG APE P1k . B.\LPA YYLR V G .CQLRNQV YEGE KK L G .PY.. KK-- 1S LL~iEXL polo LE I YEGE. KKF. L A I",XIIAILJ KK- S1I

.. c L NESE Y I MSD2 V' FPS G PI A LFiC FFTT /GL iiSRR L ELGKHSHSg] Snk YI PLEH R[R P HSi4KQK-1X -E G VI Subdomains V I -A -B VI I VIII D WS G R Plk I IN TL KMETSCL ETLRIKKNEYSIKH--IiPVAASLIQKITD TA TI ELL DEFF (305) polo M ATL E DTIDTSKIKKCEYVK-FSY - LKPAADMVEIA PNPES I LEL E|FLI (277) MSD2 F Itj4S|L!VMTLAARLTKTiLD IKYERIfKCRDF RDKPAIDDEGKIALINRDIESQLIM YVFF (335) Snk A8IOLCVMpVTbjR ETTNL ET RCREAA SS-- APAKHLAS KN D .LDII -D[W (330) Subdomains IX X XI FIG. 2. Comparison of the Plk catalytic domain to the catalytic domains of polo (11), MSD2 (A. Sugino, personal communication), and Snk (22). Identical amino acids are on a shaded background, and similar amino acids (7) are boxed. Amino acids indicated above the comparison refer to residues considered invariant or highly conserved in the serine/threonine protein kinase family (4). Subdomains refer to the conserved subdomains found in the catalytic domains of all protein kinases (4, 9). Gaps represented by dashes were introduced to maximize the alignment. Amino acid residue numbers used in this study are indicated. Amino acid alignments were carried out by using the HOMED editor of Peter Stockwell (Otago University, New Zealand).

related to Snk (serum-inducible kinase), the product of an Asn-) and subdomain Vlll (-Gly-Thr-Pro-Asn-Tyr-Ile-Ala- early response gene, identified in NIH 3T3 cells (52% iden- Pro-Glu) that suggest Plk is a serine/threonine protein kinase tity) (22), and the Saccharomyces cerevisiae cell cycle kinase rather than a tyrosine protein kinase (4, 9). The sequences in MSD2 (multiple suppressor of dbf4 2) (49% identity) (A. subdomain VlB appear to display some subfamily specific- Sugino, personal communication). ity. Plk includes a Gly next to the Leu in the sequence The sequence similarity between Plk, polo, Snk, and Asp-Leu-Lys-Leu-Gly-Asn; to date no protein kinases other MSD2 extends beyond the catalytic domain. In the C-termi- than polo, Snk, and MSD2 include a Gly immediately prox- nal extracatalytic region, Plk shares 43% identity over 288 imal to the Leu at this position. The presence of both these amino acids with polo (11), 33% identity over 166 amino acids residues may provide a marker for membership of this with MSD2, and 40% identity over 180 amino acids with Snk subfamily of protein kinases. (22). The similarity between Plk, polo, Snk, and MSD2 is Pik Expression in Fetal and Adult Mouse Tissues. We have particularly striking in one region of the noncatalytic domain determined the pattern of Plk expression by Northern blot [aa 410-439 in Plk (Fig. 3)], suggesting that these molecules analysis of mRNAs prepared from fetal, newborn, and adult share a close functional and evolutionary relationship. mouse tissues. A single mRNA species of approximately 2.2 Searches of the data base (April 1993) have revealed that no kb was detected in adult bone marrow as well as newborn and other characterized protein kinase contains this domain, adult spleen (Fig. 4). Plk mRNA was also expressed in fetal raising the possibility that it represents an important regula- liver, kidney, and brain (Fig. 5). The steady-state levels of tory region common to this class of molecules. mRNA present in liver and brain were higher in fetal tissues Analysis of Subdomains of Plk. The N-terminal catalytic at day 13 than at day 16. Plk mRNA was also detectable in domain of Plk displays most of the variant and invariant liver, brain, and kidney of newborn animals, albeit at lower residues that are the hallmarks of serine/threonine protein levels than in the corresponding fetal tissue. In none ofthese kinases (4, 9). The N-terminal region of the catalytic domain tissues taken from adult mice could we detect Plk mRNA. In of protein kinases includes the consensus motif, Gly-Xaa- contrast, Plk mRNA levels in the thymus increased during Gly-Xaa-Xaa-Gly (subdomain 1), that constitutes part of the fetal development, were highest in newborn animals, and ATP-binding site (4). The ATP-binding site ofPlk in common with polo (11), Snk (22), and MSD2 includes the related motif Gly-Xaa-Gly-Xaa-Xaa-Ala. Plk lacks the conserved O Q highly a)

Val residue located two positions to the C-terminal side of U) Gly-Xaa-Gly-Xaa-Xaa-Ala (4). Polo, Snk, and MSD2 all -Q (1) exhibit the Val-to-Cys change seen in Plk. Plk contains the C ° ;; residues within subdomain Vlb (-Asp-Leu-Lys-Leu-Gly- m0o m° za) en FIG. 4. Northern blot analysis Plk (410) :DNS V -.Ni:)S (439) 2.2kb of total RNA (10 ,g) isolated from polo (391) :DEGI FVJFUNpT (420) newborn (day 1) and adult (day 60) mouse spleen, adult blood, and MSD2 (513) WILIFNN G (542) ;TED bone marrow. The blot was hy- Snk (500) ;DHT G (530) GAPDH-e bridized with a Plk-specific cDNA probe (nt 1672-2476, Fig. 1 Lower) FIG. 3. Comparison of the amino acid sequence of the highly and rehybridized with a glyceral- 28S- conserved region within the noncatalytic domain of Plk with the * ...... dehyde-3-phosphate dehydroge- corresponding region in polo (11), MSD2, and Snk (22). For amino nase (GAPDH) probe. Ethidium- acid groupings see Fig. 2 legend. Amino acid residue numbers used 1 8S stained gels are shown to indicate in this study are indicated. amount of RNA loaded. Downloaded by guest on September 28, 2021 Biochemistry: Clay et al. Proc. Natl. Acad. Sci. USA 90 (1993) 4885

Liver Kidney Brain Thymus I ~~~~~~~~~~~~~~~~~I l~ I I 00 - -a a) a) a) 0 0 0 0 Q) (1 50 c -0 0 Co o 0 ( ci Co Co 70 -0 m ° 0 n CY) (0) ci) (0D :0 CY (D (1 -D z z Z IZ 2.2kb *,*

GAPDH-

s...... _ ,, _ **- .. 28S - _ O.. 1 8S

FIG. 5. Northern blot analysis of total RNA (10 ,g) from various mouse tissues: day 13 fetal, day 16 fetal, newborn, and adult (day 60).

were reduced substantially in adult thymus. With the excep- tion of bone marrow and spleen, the only adult tissues surveyed which express Plk mRNA were ovaries and testes (Fig. 6). In the case of ovaries, the level of Plk mRNA was higher in prepubescent tissue than in adult, while in testes, the level of Plk mRNA was higher in adult than in prepubes- cent mice. No Plk transcripts could be detected in adult heart, lung, muscle, stomach, or lymph node (data not shown).

DISCUSSION We report here the isolation and sequence of a protein kinase that is highly related to the enzyme from the Drosophila mutant polo and that we have designated Plk (polo-like kinase). To evaluate further the relationship of P1k to polo (11), we have constructed a phylogenetic tree in which the catalytic domain of P1k is compared with the catalytic do- mains of other previously described serine/threonine protein kinases (4, 9, 23, 24). As shown in Fig. 7, many of these kinases fall into subfamilies having similar primary structures and also sharing similar modes ofregulation and/or substrate specificities (4, 9). While Plk and polo appear more closely related to the SNF1 protein kinase subfamily than other serine/threonine kinases (4), they clearly form a distinct subfamily that also includes MSD2 and Snk (22). In view of Ovaries Testes 1 1 a) c)a) nn v 0oD 0 -0 0 D0 2: a) I -L a) - Z a- < L-

2.2kb 0 a..

'I I ": GAPDH- 0* 0: K1N28 a a . CDC28 28S cdc2+ FIG. 7. Phylogenetic analysis of the catalytic domains of 26 18S- serine/threonine protein kinases. The alignment was calculated according to the tree-building concept of Fitch and Margoliash (23) as implemented by Feng and Doolittle (24), using sequences from the FIG. 6. Northern blot analysis of total RNA (10 /g) from mouse data base of S. Hanks (4, 9) of the Salk Institute. Branch order is a testes and ovaries: newborn, prepubescent (day 20), and adult (day function of structural similarity; branch length indicates sequence 60). identity. Downloaded by guest on September 28, 2021 4886 Biochemistry: Clay et al. Proc. Natl. Acad. Sci. USA 90 (1993)

the similar overall structure and the relatedness displayed expression of Plk by in situ hybridization as well as to study between Plk and polo outside the catalytic domain, we its expression during the mammalian cell cycle. believe that Plk is the murine homologue ofDrosophila polo. Our original goal was to identify protein kinases that might We thank Drs. M. Ernst and E. Stanley for comments on the serve a unique role in some aspect of growth regulation in manuscript, C. Backus for typing the manuscript, G.-F. Tu for help primitive hemopoietic cells. As Plk sequences were originally with sequencing, Drs. H. Reid and 0. Bernard for the neuroepithelial isolated from a population of primitive hemopoietic cells, it cDNA library, D. Grail for help with animals, C. Buckmaster for the ICR CD1 genomic DNA, and Dr. Akio Sugino for making available is not surprising to see the expression of Plk in hemopoietic results prior to tissues such as bone marrow, spleen, and thymus. However, publication. expression was not restricted to these tissues, although the 1. Norbury, C. & Nurse, P. (1992) Annu. Rev. Biochem. 61, patterns of Plk expression that we have observed suggest an 441-470. association with rapidly dividing tissues. This conclusion is 2. Jacobs, T. J. (1992) Dev. Biol. 153, 1-15. drawn principally from the dramatic differences in steady- 3. Pelech, S. L. & Sanghera, J. S. (1992) Trends Biochem. 17, state levels of Plk mRNA in fetal tissues compared with their 233-238. adult 4. Hanks, S. K., Quinn, A. M. & Hunter, T. (1988) Science 241, counterparts. Most strikingly, while Plk is expressed in 42-52. fetal liver, kidney, and brain, no Plk mRNA was detected in 5. Hunter, T. (1987) Cell 50, 823-829. the corresponding adult tissues. While we cannot rule out an 6. Lindberg, R. A., Quinn, A. M. & Hunter, T. (1992) Trends essential functional or morphogenetic role for Plk during Biochem. 17, 114-119. development, its expression in a range of fetal tissues may 7. Letwin, K., Mizzen, L., Motro, B., Ben-David, Y., Bernstein, simply reflect active cellular proliferation. This notion is A. & Pawson, T. (1992) EMBO J. 11, 3521-3531. consistent with the established function of polo, which in 8. Kemp, B. E. & Pearson, R. B. (1990) Trends Biochem. 15, normal Drosophila is expressed in tissues undergoing active 342-346. 9. Hanks, S. K. & Quinn, A. M. (1991) Methods Enzymol. 200, mitosis. In larvae homozygous for polo mutation the diploid 38-62. imaginal cells are blocked in mitotic division and so become 10. Wilks, A. F. (1989) Proc. Natl. Acad. Sci. USA 86, 1603-1607. unable to proliferate to become adult structures (11, 21). 11. Llamazares, S., Moreira, A., Tavares, A., Girdham, C., An intriguing observation to emerge from our analysis is Spruce, B. A., Gonzalez, C., Karess, R. E., Glover, D. M. & that, with the exception ofhemopoietic tissues, the only adult Sunkel, C. E. (1991) Genes Dev. 5, 2153-2165. tissues in which we have detected Plk expression are ovaries 12. Bertoncello, I., Bradley, T. R. & Hodgson, G. S. (1989) Exp. and testes. In the female, oocytes are stored in primordial Hematol. 17, 171-176. follicles, which initiate growth at any time from birth to the 13. Bertoncello, I., Bradley, T. R. & Watt, S. M. (1991) Exp. end offertile life (25). Oocytes enter growth phase around the Hematol. 19, 95-100. day of birth 14. Bartelmez, S. H., Bradley, T. R., Bertoncello, I., Mochizuki, and reach full maturity around day 15 (25). The D. Y., Tushinski, R. J., Stanley, E. R., Hapel, A. J., Young, oocytes are surrounded by granulosa cells, which undergo I. G., Kreigler, A. B. & Hodgson, G. S. (1989) Exp. Hematol. rapid proliferation in response to hormonal stimulation (25, 17, 240-245. 26). While we have detected Plk transcripts in ovaries at 15. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular various stages of development, the levels varied depending Cloning: A Laboratory Manual (Cold Spring Harbor Lab., on the age of the animal. Most strikingly, Plk transcripts were Plainview, NY). present at low levels in the ovaries of newborn mice, were 16. Reid, H. H., Wilks, A. F. & Bernard, 0. (1990) Proc. Natl. dramatically elevated in prepubescent mice, and decreased Acad. Sci. USA 87, 1596-1600. thereafter. On the basis of Northern 17. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. analysis we cannot Acad. Sci. USA 74, 5463-5467. distinguish between expression of Plk in the expanding 18. Pearson, W. R. & Lipman, D. J. (1988) Proc. Natl. Acad. Sci. population ofoocytes or the proliferating granulosa cells. The USA 85, 2444-2448. pattern of expression of Plk is, however, reminiscent of that 19. Chomczynski, P. & Sacchi, N. (1987) Anal. Biochem. 162, described for c-kit (27) and c-mos (26, 28), which show an 156-159. increase in expression corresponding to maximal oocyte 20. Kozak, M. (1991) J. Cell Biol. 115, 887-903. growth. In the male, the testes consist of hormonally respon- 21. Sunkel, C. E. & Glover, D. M. (1988) J. Cell. Sci. 89, 25-38. sive somatic cells (the Sertoli and Leydig cells) as well as a 22. Simmons, D. L., Neel, B. G., Stevens, R., Evett, G. & Erik- small number of self-renewing stem cells that serve as the son, R. L. (1992) Mol. Cell. Biol. 12, 4164-4169. progenitors of a range of spermatogenic cells, including 23. Fitch, W. M. & Margoliash, E. (1967) Science 155, 279-284. mature 24. Feng, D.-F. & Doolittle, R. F. (1987) J. Mol. Evol. 25, 351-360. spermatozoa (29, 30). Our observation that the high- 25. Bacaharova, R. (1985) in Developmental Biology, ed. Browder, est levels of Plk expression are associated with adult testes L. W. (Plenum, New York), Vol. 1, pp. 453-523. probably reflects active spermatogenesis (26, 29) and the fact 26. Goldman, D. S., Kiessling, A. A., Millette, C. F. & Cooper, that Sertoli and Leydig cells constitute a smaller proportion G. M. (1987) Proc. Natl. Acad. Sci. USA 84, 4509-4513. of total cell numbers in adult animals than at earlier times 27. Manova, K., Nocka, K., Besmer, P. & Bacharova, R. F. (1990) (30). It will be interesting to establish the patterns of Plk Development 110, 1057-1069. expression in mutant mice that exhibit defects in germ cell 28. Mutter, G. L. & Wolgemuth, D. (1987) Proc. Natl. Acad. Sci. development (27, 31). USA 84, 5301-5305. In summary, we have described the isolation of a mam- 29. Bellve, A. R., Cavicchia, J. C., Millette, C. F., O'Brien, D. A., Bhatnagar, Y. M. & Dym, M. (1977) J. Cell Biol. 74, malian protein kinase that is likely to be the murine homo- 68-85. logue of Drosophila polo. The patterns of Plk expression 30. Willison, K. & Ashworth, A. (1987) Trends Genet. 3, 351-355. suggest an association with some aspect of cellular prolifer- 31. Bennett, W. I., Gall, A. M., Southard, J. L. & Sidman, R. L. ation. It now remains to more precisely establish the sites of (1971) Biol. Reprod. 5, 30-58. Downloaded by guest on September 28, 2021