Vol. 10, No. 7MOLECULAR AND CELLULAR BIOLOGY, JUlY 1990, p. 3578-35830270-7306/90/073578-06$02.00/0Copyright C) 1990, American Society for Microbiology

Developmentally Regulated Protein-Tyrosine Kinase Genesin Dictyostelium discoideumJOHN L. TAN AND JAMES A. SPUDICH*

Departments of Cell Biology and Developmental Biology, Stanford University School of Medicine,Stanford, California 94305

Received 5 February 1990/Accepted 20 April 1990

Dictyostelium discoideum, an organism that undergoes development and that is amenable to biochemical andmolecular genetic approaches, is an attractive model organism with which to study the role of tyrosinephosphorylation in cell-cell communication. We report the presence of protein-tyrosine kinase genes in D.discoideum. Screening of a Dictyostelium cDNA expression library with an anti-phosphotyrosine antibodyidentifies fusion proteins that exhibit protein-tyrosine kinase activity. Two distinct cDNAs were identified andisolated. Though highly homologous to protein kinases in general, these kinases do not exhibit many of thehallmarks of protein-tyrosine kinases of higher eucaryotes. In addition, these genes are developmentallyregulated, which suggests a role for tyrosine phosphorylation in controlling Dictyostelium development.

The coordination of cell cycle, growth, and differentiationin multicellular organisms depends on a complex network ofsignals, receptors, and transducing elements. The unregu-lated proliferation of transformed cells results from an un-coupling of growth control from cell-cell communication.Protein-tyrosine kinases are integral components of thetransduction pathway of receptors for growth factors, suchas insulin and epidermal growth factor (23, 24), and areencoded by a major class of viral oncogenes (9).Dictyostelium discoideum is an organism particularly

suited to biochemical and genetic studies (19). The discoveryof efficient gene targeting by homologous recombination (4)has made D. discoideum a practical system for the study ofgene and protein function. Unlike many unicellular organ-isms, D. discoideum has a complex developmental program.In response to limited nutrients, these cells aggregate anddifferentiate to form a multicellular pseudoplasmodium.These characteristics make D. discoideum an attractiveorganism with which to study the effects of tyrosine phos-phorylation on various cellular functions. We report theisolation of two cDNA clones that encode protein-tyrosinekinases from a Dictyostelium expression library.

MATERIALS AND METHODSScreening and isolation of cDNA clones. A Dictyostelium

cDNA library (generously provided by Peter Devreotes,Johns Hopkins University, Baltimore, Md.), constructed inAgtll from poly(A)+ mRNA which was isolated from cellsthat were placed in a medium with limited nutrient supply for4 h to induce development, was screened with the anti-phosphotyrosine monoclonal antibody PY20 (ICN Biochem-icals). The library was plated at a density of -30,000 plaquesper 150-mm LB agar plate and induced with IPTG (isopro-pyl-,-D-thiogalactopyranoside)-impregnated nitrocellulosefilters for 4 h at 37°C. A total of -300,000 plaques wasscreened. The nitrocellulose filters were blocked with 20%fetal calf serum in TBS (50 mM Tris hydrochloride [pH 7.5],150 mM NaCl) and incubated with 0.5 ,ug of PY20 per ml inTBS containing 20% fetal calf serum. Alkaline phosphatase-conjugated goat anti-mouse secondary antibody (Bio-RadLaboratories) and 5-bromo-4-chloro-3-indolyl phosphate

* Corresponding author.

(BCIP) were used to detect plaques recognized by theanti-phosphotyrosine antibody. Positive plaques were puri-fied by three additional screenings until all plaques in thescreen were stained with the anti-phosphotyrosine antibody.

Bacterial expression of cDNAs. The isolated cDNAs werecloned into the EcoRI site of the bacterial expression plas-mid pGEX1 (17) and transformed into Escherichia coliDH5o. Bacteria harboring the appropriate plasmid weregrown to an optical density at 650 nm of 0.6 and were eithergrown for an additional 2 h (untreated) or treated with 1 mMIPTG for 2 h at 37°C.Phosphoamino acid analysis. Bacteria harboring the appro-

priate expression plasmid were labeled in vivo with 32p; in Lbroth as previously described (21). Lysates of labeled bac-teria corresponding to 100 ,ul of a culture with an opticaldensity at 650 nm of 0.6 were loaded per lane on a 12%sodium dodecyl sulfate-polyacrylamide gel. Proteins wereresolved by electrophoresis and transferred onto polyvinyl-idene difluoride membranes (Immobilon; Waters Associates,Inc.). Blots were analyzed with the PY20 antibody. Highlyreactive bands were cut out, partially acid hydrolyzed, andsubjected to two-dimensional electrophoresis along withphosphoamino acid standards as previously described (3).

Sequencing of cDNA clones. The cDNAs were subclonedinto the plasmid pTZ18R (Pharmacia). Nested deletionswere generated with exonuclease III (Erase-a-base; PromegaCorp.). DNA sequencing was done on both strands bypreviously described methods (11). Sequence analyses weredone by using the University of Wisconsin Genetics Com-puter Group software packages (5).

Southern blot analysis. Dictyostelium genomic DNA wasisolated as previously described (15). Restriction enzyme-digested DNA was separated on 0.6% agarose gels andprocessed by standard methods (14). The cDNAs wereradiolabeled by random-primed synthesis and used asprobes. Hybridized blots were washed three times for 15 mineach in 0.2x SSC buffer (lx SSC is 0.15 M NaCl plus 0.015M sodium citrate) containing 0.5% sodium dodecyl sulfate at680C.

Northern (RNA) blot analysis. Dictyostelium RNA wasisolated and processed as previously described (2). Hybrid-ization and wash conditions were as described above.

3578

DICTYOSTELIUM PROTEIN-TYROSINE KINASES 3579

A B C

-+ . + - +

"ta(% J T

y30-

FIG. 1. Western immunoblot analysis of bacterial lysates probedwith anti-phosphotyrosine antibody. (A) Bacteria transformed withthe parent plasmid, pGEX1. (B) Bacteria transformed with plasmidcontaining the DPYKI insert. (C) Bacteria transformed with plasmidcontaining the DPYK2 insert. Bacteria were either untreated (-) orinduced with IPTG (+). Bacteria from 1 ml of culture were har-vested and suspended in 100 ,ul of gel-loading buffer, of which 10 [lIwas loaded per lane. Bacterial proteins were resolved by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis and transferredonto nitrocellulose. The blot was probed with the monoclonalanti-phosphotyrosine antibody PY20 and developed with horserad-ish peroxidase-conjugated goat anti-mouse antibody and 4-chloro-1-naphthol. Molecular masses are indicated to the left of the gel inkilodaltons (kD).

RESULTS AND DISCUSSION

A cDNA expression library from developing Dictyosteli-um cells was screened with an anti-phosphotyrosine anti-body. This method, which has been used to identify protein-tyrosine kinase genes in other cells (10, 13), is designed todetect expressed fusion proteins that phosphorylate tyrosineresidues. Of -300,000 clones screened, 20 reacted positivelywith the antibody probe. Seven of these clones were purifiedand categorized into two groups by cross-hybridization. Onegroup contained a 1.1-kilobase insert (DPYKI), and theother contained a 1.3-kilobase insert (DPYK2).To confirm protein-tyrosine kinase activity, the cDNA

inserts were subcloned into a bacterial expression plasmid.Western immunoblot analysis demonstrated that lysates ofbacteria expressing the fusion protein exhibit protein-ty-rosine kinase activity, as evidenced by the appearance ofmultiple proteins that reacted with the anti-phosphotyrosineantibody PY20 (Fig. 1). The difference in the bacterialproteins that are phosphorylated by DPYK1 and DPYK2presumably reflects the preferred substrate specificities ofthe two kinases. The tyrosine specificity of the Dictyostel-ium kinases was confirmed by phosphoamino acid analysis(Fig. 2).DNA sequence analysis of DPYKI and DPYK2 (Fig. 3)

S 0

Sb4kf

% TI,y

FIG. 2. Phosphoamino acid analysis of proteins from bacteriaexpressing the Dictyostelium kinases. Dashed lines represent inter-nal standards: phosphoserine (S), phosphothreonine (T), and phos-photyrosine (Y). Several proteins from IPTG-induced bacteria la-beled with 32p; were analyzed and demonstrated to containphosphotyrosine. Phosphoamino acid analysis of the most reactiveproteins in gel lanes B+ and C+ of Fig. 1 is shown.

revealed that each contains a single open reading frame thatexhibits the strongly biased codon usage typical of othercharacterized Dictyostelium genes (22). Neither of the in-serts represents a full-length transcript.Hanks et al. (7) designated within the catalytic domain of

protein kinases 11 subdomains that contain consensus se-quences thought to encode residues essential to phospho-transferase activity. Each of these subdomains is present inthe deduced amino acid sequences of the Dictyosteliumprotein-tyrosine kinases (Fig. 4). Two regions, one in sub-domain VI and one in subdomain VIII, contain sequencesthat were proposed to define hydroxyamino acid specificity(7). Although the Dictyostelium genes encode these regions,the sequences do not closely match those of protein-tyrosinekinases of higher eucaryotes (Fig. 4). The divergence of theDictyostelium sequences from highly conserved protein-tyrosine kinase sequences demonstrates that these sequencemotifs are not absolutely required for tyrosine specificity.Nonetheless, a single tryptophan at position 273 (Fig. 4) is

kD

DPYKI

94-

67-

42-

sS _

14-

-p~ m- '0.

DPYK2

VOL. 10, 1990

3580 TAN AND SPUDICH MOL. CELL. BIOL.

A1

91

181

271

361

452

542

632

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812

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GAATTCCGCCCATTTGGTGGTTGGGAAACTCAATCATCATTATCACATCCACCATCACGTCCACCACCACCTCCACCACCACCACCACAAR P F G G W E T Q S S L S H P P S R P P P P P P P P P Q

CTACCAGTrAGATCAGAATACGAGA¶GATrTCAATGAATFAGAATNGTCAACCATrGGTAAAGGTTTCTTTGGTGAAGTAAAGAGAL P V R S E Y E I D F N E L E F G Q T I G K G F F G E V K R

GGT1~AT?GG&GAGAGAcGATGTNCCATAAAAATC&TCTATcGTGATCAATPCAAAACCAAATC&TCA~1¶GGTTATGTTTCAAAATGAAG Y W R E T D V A I K I I Y R D Q F K T K S S L V N F Q N E

GTTGGAATACTAAGTAAACAAAGACATCCAAATGTACATTGGTGCATGTACTGC&GGAGGTGAAGATCATCATTGTATAGTAV G I L S K L R H P N V V Q F L G A C T A G G E D H H C I V

ACAGAATGGATGGGTGGAGGTAGTTTAAGACAGTTCTTGACTGATCATTTCAATTTACTCGAACAAAATCCACATATTCGTGAAGTTGT E W N G G G S L R Q F L T D H F N L L E Q N P H I R L K L

GCTrGTATTGCAAGGAATGAATTATC TACATGG TTGGACTCCACCCATTCTTCATCGTGACTTATCCTCAAGAAACATTTTATTGA L D I A K G M N Y L H G W T P P I L H R D L S S R N I L L

GATCACAACATCGATCCAAAGAATCCGTTAGTTTCCTCAAGACAAGATATTAAATGTAAGATC CTGATCGTCTAAGTAGATTAAAGD H N I D P K N P L V S S R Q D I K C K I S D F G L S R L K

AAGGAACAAGCCTCTC&AATGAC CAATCGGTTGG1STGTATTCCCTACATGGCACCAGAGGTSTTCAAAGGCGATAGTAATAGTGAAAAGK E Q A S Q M T Q S V G C I P Y N A P E V F K G D S N S E K

AGTGATT GA CTCTGATGAACCTCAACAAGATATGAAACCAATGAAAATGGCTCACS D V Y S Y G H V L F E L L T S D E P Q Q D N K P M K N A H

TTGGCTGCCTATGAATC TATCGTCCTCCAATTCCATTAACTACCTCTTCCAAGTGGAMATAACTCAAGrGGA !AL A A T E S Y R P P I P L T T S S K W K E I L T Q C W D S N

CCTGATAGTCGTCCAACCAACAAATCATGTTCATCTCAAAGAAATGGAAGATCAAGGTGTATCTTC TT TGCATCTGTACCTGTTP D S R P T F K Q I I V H L K E M E D Q G V S S F A S V P V

CAAACTATTGATACTGGTGTTTATGCPTAATTlTITl¶TTTATAATA A CAAAACAAAAAAAAATAATAATAAATAQ T I D T G V Y A *

1082 TAATCACTTCAACTCGGAATTC 1103

B1 GAAT?CCGATTCTACAATACAAAACTCTACTAAAGATATCACATTTTTAGTTTGTGATAATCCTGATTCAACTAAAGAAAAGAGTAAC

R F Y N T T N S T K D I T F L V C D N P D S T K E K S N

91 G?ACAATACTTCATCAATAATTTCCGCTTCAAATTTAAATAGACATATAACACCAAATTCTCATATGAGACCTAGAGGTAGATCAATTV S N T S S I I S A S N L N R H I T P N S H M R P R G R S I

181 TCTGAATCTTTAGTTATGTAAAS E S L I N S P I N K E S L N D I Q R A I E S E K I K K T K

271 TATF E E L K S I L G E R E Y I I D I N D I Q F I Q K V G E G A

361 = _TGATNG _GGTATTCATGlrCCATAAAAAAGTTAAAGATrATAGGAGATGAAGAACAATTCAAAGAGF S E V W E G W W K G I H V A I K K L K I I G D E E Q F K E

452 G AA TGTTTATGG5SNTGTTATWCCAGCATGTATCR F I R E V Q N L K K G N H Q N I V H F I G A C Y K P A C I

542 ATAACAGAGTATATGGCAGGTGGTAGTTAECATTTArCATAATCCATT AGTTAAATA CCAI T E Y H A G G S L Y N I L H N P N S S T P K V K Y S F P L

GTTTTGAAAATGGCAACCGACV L R M A T D

CTATTGGATGAATTGGGTAAL L D E L G N

GGCATTTGCAATCCAAGATGGG I C N P R W

TGGCAGGGCTATTACATCCTTCCATCAC CA MAACCAGCA TN A L G L L H L H S

ATAAAGATCTTCCI K I S D F G L S A

AGACCACCCGAATIGACAAAGAATTTAGG7R P P E L T K N L G

TG

TC

I T I V H R D L T S Q N I

E K S R E G S N T M T N G

CACTACCGGAAAAGGIATGTTA CTCTTAH Y S E K V D V Y C F S L

GTAGTTTGGGAAATTTTAACTGGCGAATTCC TCTCTGATTTAGATGGATCTCAACGATCCGTCAAGTAGCTTATGCTGGTTTAAGAV V W E I L T G E I P F S D L D G S Q R S A Q V A Y A G L R

CCA GCTCTTAACTCAATGTTGAGACGGCTGATCCAAATGATAGACCTCCCTTTACCP P I P E Y C D P E L K L L L T Q C W E A D P N D R P P F T

1082 TATATAGTAAACAAATTAAAAGAAATCTCTTGGAATAATCCAATTGGTTTCGTCTCTGATCAATTCTATCAATATAGCGAACCTTCAACTY I V N K L K E I S W N N P I G F V S D Q F Y Q Y S E P S T

1172 CCAAGATTAGCATTATCAAATCAATCTTCAAATTCAAGTAGTATTTCTTTATCACCAACTAAATTATAAAAA&AAAAAAAAAAAAAAACAP R L A L S N Q S S N S S S I S L S P T K L *

1262 AATTTCAAACACCAAACACCACCACTCATCAAAATCGoAATTC 1304

FIG. 3. Nucleotide and deduced amino acid sequences of DPYK1 (A) and DPYK2 (B). The deduced amino acid sequences are shownbelow the corresponding nucleotide sequences. The single-letter amino acid code is used. The EcoRI sites flanking the clones are shown inboldface type. Stop codons are denoted with asterisks.

632

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rrm

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DICTYOSTELIUM PROTEIN-TYROSINE KINASES 3581

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DPYK1 NELEFGQTI FG RGYWRE ........ TD IYRDQFKTKSS

DPYK2 NDIQFIQK FS EGWWKG........ I LKIIGD.EECQFFps EDVILGERI FG SGRLRA... NT..DNTP CRETLPPE. L

Ros DKLNLHKL FG EGTALDILADGSGESR 4YTLKRGAT. .DQESrc ESIRLEAK FG JMTWND...HE....TTR LKPGTM.... S

cAPK-a DQFERIKT FG LVKHME.......1TGNH LDKQKVVKLKQ

I

A1 2 3 4 5 6 7

B1 2 3 4 5 6 7

II91

DPYK1 LVMFQ GILSKLRHPNVVQFLGACTAGGEDHHCIVTEWMGGGSLRQFLDPYK2 KERF I NLKKGNHNIVNFIGACYK .... PACIITEYMAGGSLYN IL

Fps KAKFL RILK CNHPNIVRLIGVCTQ. . KQPIYIVMELVQGGDFLSFLRos KSEFL HLMS FDHPHILKLLGVCLL. . NEPQYLILELMEGGDLLS YLSrc PEAFL QVMKKLRHEKLVQLYAVVSE ... EP YIVIEYMSKGSLLDFL

cAPK-a IEHTL KRILQAVNFPFLVKLEFSFKDN. . SNLYMVMEYVPGGEMFSHL

III IV V166 171

DPYKI TDHFNLLE.... QNPHIRLKLALDIAKGMNYLHGWTPPILH SSI;ILDPYK2 HNPNSSTPK.VKYSFPLVLKMATDMALGLLHLHSI..TIVH TS IL

Fps RSKGPRLK...... MKKLIKMMENAAAGMEYLESK .. HCIH LAAFLRos RGARKQKFQSPLLTLTDLLDICLDICKGCVYLEKM..RFIH AAF LSrc KGEMGKYL.....RLPQ LVDMAAIASGMAYVERM ..NYVH RA L

cAPK-a RRIGRFSE....... PHARFYAA IVLTFEYLHSL.. DLIY IKPE L

VI

DPYKIDPYK2

FpsRosSrc

cAPK-a

184 1 5 186 10

LDHNIDPKNPLVSSRQDIKCKI F SRLKKEQASQNT..QSVGCIPYMLDELG.NIKI SAEKSREGSNTMT NGG ICNPRWR

VTEKN.TLKI SRQEEDGVYASTGGMKQIPVKWTVSEKQ YGSCSRVVKI F LARDIYKNDYYRKRGEGLLPVRWMVGENL.VCKV FARLIEDNEYTAR.QGAKFPIKWTIDQQG. YIQV FAKR.VKGRTWTLC... GTPEYL

VII

kb

10- 0'

6-4-

3-

2- a

1.6-

1-VIII

208 220 225

DPYK1 A F.KGDSNSEKSOVYS VLFELLTSDEPQQDM.... KPMKMAHLAADPYK2 PP .LTKNLGHYSEK' YC LVVWEILTG.EIPFSDL. .DGSQRSAQVA

Fps AP L.NYGWYSSE IWSFILLWEAFSLGAVPYANLSNQQTREAIEQGRos A L.IDGVFTNH WAF LVWETLTLGQPYPGLSNI EVLHHVRSGSrc A A.LYGRFTIK WS ILLTELTTKGVPYPGMVNREVLDQVERG

cAPK-a AP I.LSKGYNKA WALLIYEMAA.GYPPFFADQPIQIYEKIVSG

Ix x273 260b 298

DPYK1 YESYRPPIPLTTSSKWKEILTQCWDSNPD TFKQIIVHLKEMEDQGVDPYK2 YAGLRPPIPEYCDPELKLLLT CWEADPN PFTYIVNKLKEISWNNP

Fps VRLEPPEQ ... CPEDVYRLMQ CWEYDPH SFGAVHQDLIAIRKRHRRos GRLESPNN CPDDIRDLMTRCWAQDPH TFFYI QHKLQEIRHSPLSrc YRMPCPPE ... .CPESLHDLMCQCWRKDPE TFKYL A LLPACVLEV

cAPK-a .KVRFPSH. . FSSDLKDLLRNLLQVDLT GNLKD V DIKNHKWFA

XI

0.5-

FIG. 5. Southern blot analysis of Dictyostelium genomic DNA.Autoradiograms of blots probed with DPYKJ (A) and DPYK2 (B)are shown. Lanes: 1, BamHI; 2, BclI; 3, ClaI; 4, EcoRI; 5, EcoRV;6, HindIII; 7, PstI. Sizes of DNA bands are given at the left inkilobases (kb).

FIG. 4. Amino acid sequence alignment of protein kinase cata-lytic domains. Alignment follows that of Hanks et al. (7). Numberingof the amino acids in the catalytic domain is with respect to thecyclic AMP-dependent protein kinase (cAPK-a) sequence. Amongprotein-tyrosine kinases, Ros and Fps show the highest homology toDPYK1 and DPYK2, respectively. The Src and cAPK-a sequencesare included as prototypical protein-tyrosine and protein-serine/threonine kinase sequences, respectively. Boxed residues indicatethose amino acids conserved in 62 of 65 protein kinase sequencescompared by Hanks et al. (7). The solid circle indicates the positionof the tyrosine residue conserved in protein-tyrosine kinases ofhigher eucaryotes. The arrowhead indicates the tryptophan con-served in protein-tyrosine kinases, including the Dictyosteliumclones. Roman numerals below the sequences indicate conservedkinase subdomains designated by Hanks et al. (7). The single-letteramino acid code is used. The sequences of Fps (TVFVF), Ros(TVFVUR), Src (TVFVR), and cAPK-a (OKB02C) were obtainedfrom the National Biomedical Research Foundation database withthe call codes given in parentheses.

present in all protein-tyrosine kinases, including the Dicty-ostelium kinases, but is conspicuously absent in the protein-serine/threonine kinases, with the exception of Mos (7).

In a comparison of known protein kinase sequences (7), 15amino acid residues specifically positioned within the cata-lytic domain were found to be conserved in 62 of the 65compared sequences. Each of these residues is present at the

analogous position in both Dictyostelium genes, with theexception of a single serine-for-glycine substitution inDPYK2 at position 225 (Fig. 4).A tyrosine residue corresponding to the major autophos-

phorylation site in pp60-vsrc is conserved in all protein-tyrosine kinases of higher eucaryotes (9, 23). In protein-serine/threonine kinases, seine or threonine is present at theanalogous site. Interestingly, neither DPYKI nor DPYK2encodes tyrosine residues within this region. Rather, likeseveral protein-serine/threonine kinases, both Dictyosteliumclones encode seine. This suggests that the Dictyosteliumprotein-tyrosine kinases may be evolutionary hybrids, ex-hibiting motifs of both tyrosine- and serine/threonine-spe-cific kinases.A comparison of the Dictyostelium sequences with those

in the National Biomedical Research Foundation database(release 18.0, September 1988) reveals high homology withboth protein-tyrosine and protein-serine/threonine kinases.DPYK1 was found to be most homologous to the kinase-related transforming proteins Mos and Ros, sharing 28%identity over 306 amino acids and 28% identity over 260amino acids, respectively. DPYK2 was found to be mosthomologous to the kinase-related transforming proteins Fpsand Raf-a, sharing 28% identity over 316 amino acids and31% identity over 270 amino acids, respectively. The homol-

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molecular genetic approaches in a study of signal transduc-tion mechanisms during development.

ACKNOWLEDGMENTS

We thank Aled Edwards for suggesting the approach used hereand for critical discussions. We also thank Rex Chisholm forNorthern blots, John Bell and Rick Lindberg for reagents andprotocols, Agnieszka Rawa and Clea Chandmal for expert technicalassistance, and the investigators in the laboratory of J. A. Spudichfor helpful discussions.

This research was supported by a Public Health Service grantfrom the National Institutes of Health to J.A.S.

FIG. 6. Northern blot analysis of Dictyostelium cells growingvegetatively (V) or after 4 h of development (4h). Ten micrograms oftotal Dictyostelium RNA was loaded per lane.

ogies to Mos and Raf-a are striking in that among theprotein-serine/threonine kinases, Mos and Raf-a are phylo-genetically closest to the protein-tyrosine kinases (7). TheDictyostelium protein-tyrosine kinases thus have the prop-

erties of structural mosaics (12), in that they possess ele-ments of both protein-tyrosine and protein-serine/threoninekinases.Although protein-tyrosine kinase activity has been dem-

onstrated in Rhodospirillum rubrum (20), Saccharomycescerevisiae (16), Schizosaccharomyces pombe (6), and Strep-tococcus pyogenes (1), DPYK1 and DPYK2 are the firstcharacterized protein-tyrosine kinase genes from a unicellu-lar system. Since D. discoideum, a cellular slime mold, isthought to have diverged from the mammalian branch earlierin evolution that the ciliates (18), the identification of pro-tein-tyrosine kinase genes in this organism argues againsttheir proposed recent evolutionary development. The struc-tural mosaic nature of the Dictyostelium protein-tyrosinekinases is consistent with the hypothesis that the catalyticdomains of these enzymes are derived from mutations of anarchetypal protein-serine/threonine kinase domain (7, 8).

Southern blot analysis (Fig. 5) of DPYKI and DPYK2suggests that both genes exist as a single copy in D.discoideum. However, in lower-stringency screenings (2x

SSC washes), DPYKJ appeared to hybridize to multiplebands, suggesting the presence of several highly homologousgenes. In contrast, DPYK2 did not appear to hybridize toother bands under these conditions (data not shown).Both DPYKJ and DPYK2 appear to be developmentally

regulated genes. Northern (RNA) blot analysis showedincreases in the levels of both transcripts as D. discoideuminitiated its developmental cycle (Fig. 6). In addition, theDPYKJ probe appeared to hybridize to a number of RNAbands, consistent with the possibility that DPYKI is amember of a multigene family.The presence of developmentally regulated protein-ty-

rosine kinases in D. discoideum, an organism that reliesupon intercellular signals to control growth and develop-ment, is consistent with the hypothesis that protein-tyrosinekinases act as transducing elements in cell-cell communica-tion and provides the opportunity to exploit biochemical and

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