Proc. Nati. Acad. Sci. USA Vol. 91, pp. 2597-2601, March 1994 Biochemistry Ctk: A - related to Csk that defines an family SABINE KLAGES*, DIETER ADAM*, KENNETH CLASS, JOSEPH FARGNOLI, JOSEPH B. BOLEN, AND ROBERT C. PENHALLOWt Department of Molecular Biology, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543 Communicated by Peter M. Howley, December 23, 1993 (receivedfor review December 15, 1993)

ABSTRACT We used the polymerase chain reaction with Here we report the molecular cloning and preliminary degenerate oligonucleotide primers to search for Csk-related functional characterization of a nonreceptor PTK that is kinass. A cDNA coding for a Csk-like protein-. related to Csk.J This PTK, designated Ctk for Qsk-type was cloned from mouse brain and was designated ctk, for protein-tyrosine kinase, was found to be a 52-kDa protein gcsk-bype protein-tyrosine kinase. The 1.9-kb ctk mRNA was expressed primarily in brain and is predicted to be structur- found to be expressed predominantly in brain and capable of ally similar to Csk. Our results indicate that like Csk, Ctk can encoding a 52-kDa protein-tyrosine kinase. The amino acid phosphorylate members of the Src family of PTKs at the sequence of Ctk was found to possess 53% identity with mouse regulatory tyrosine residue. Thus Ctk and Csk define a family Csk, shared all the predicted structural features of Csk, and of that phosphorylate carboxyl-terminal regulatory was capable of phosphorylating the carboxyl-terminal con- tyrosine residues. served tyrosine of Src family members. Our results thereby indicate that ctk represents a gene that defines a family of AND METHODS structurafly and functionally related Csk-ilke protein-tyrosine MATERIALS kinae. PCR Amplification and cDNA Isolation. The murine ctk cDNA was isolated from a mouse whole-brain cDNA library The src family of protooncogenes represents a group of by PCR amplification ofphage DNA using nested degenerate nonreceptor protein-tyrosine kinases (PTKs) that currently oligonucleotide primers and Taq polymerase (15). Primer 1 contains nine members: Blk, Fgr, Fyn, Hck, Lck, Lyn, Src, [5'-GT(G/A/T/C)-CA(C/T)-AG(G/A)-GA(C/T)-TT(G/A)- Yes, and Yrk (reviewed in refs. 1 and 2). Enzymatic activa- GC-3'] coded for the amino acid sequence VHRDLA; primer tion of Src family PTKs has been identified as an important 2 [5'-CC-(G/A)AA-(G/A/T/C)(G/C)(A/T)-CCA-(G/A/T/ step in a number of signal transduction pathways in a wide C)AC-(G/A)TC-3'] carried the reverse complement of the variety of types, as well as a critical event in stimulating coding sequence for the amino acids DVWSFG, and primer the oncogenic potential of this family of (reviewed 3 [5'-GGC-(C/T)TC-(G/A/T/C)GG-(G/A/T/C)GC-(G/A/ in refs. 1-4). The enzyme activity of Src family PTKs is T/C)GT-CC-3'] carried the reverse complement ofthe coding primarily regulated through the of a con- sequence for the amino acids WTAPEA, located upstream of served carboxyl-terminal tyrosine residue (1-4). Phosphor- DVWSFG. The phage DNA was initially amplified with ylation of this residue reduces kinase activity, while dephos- primers 1 and 2. The -200-nt products were isolated and phorylation by protein-tyrosine-phosphatases results in ele- subjected to a second round of PCR using primers 1 and 3. vated Src family activity (1-4). Mutations that The reaction products were cloned by using the TA cloning change this conserved tyrosine residue to some other amino (Invitrogen) and sequenced. The ctk PCR fragment was acid or deletions that remove this region of the protein result purified, labeled with [a-32P]dCTP and Klenow polymerase in the constitutive activation of Src family enzymes and using random primers, and used to probe a mouse whole promote their potential oncogenic functions (1-4). brain phage library. About 5 x 106 phages were screened and Under normal physiologic conditions, phosphorylation of positive clones were isolated by two additional rounds of the regulatory tyrosine residue appears to involve the activity screening. The cDNA inserts were rescued as pBluescript of PTKs distinct from members of the Src family. To date, (Stratagene) clones by in vivo excision and their sequences tyrosine phosphorylation of Src family PTKs has been at- were determined. Analysis of RNA. Total cellular RNA was extracted by tributed to the activity of a recently cloned 50-kDa cytoplas- standard procedures (16), fractionated in 1% agarose/ mic PTK designated Csk (for carboxyl-terminal Src kinase). formaldehyde/Mops gels, transferred in 20x standard saline In addition to a PTK catalytic domain, Csk possesses one Src citrate (SSC) by capillary blotting to Hybond-N membranes homology 3 (SH3) and one SH2 domain but lacks a defined (Amersham), and probed with gel-purified 32P-labeled ctk, autophosphorylation site, a carboxyl-terminal regulatory ty- csk, or f3-actin probes (17). For dot blots, the RNA samples rosine phosphorylation site, and an amino-terminal myris- were loaded in 50%o formamide/2.2 M formaldehyde/20 mM toylation site that are features of Src family PTKs (5-8). Mops/4x SSC and washed twice in 1Ox SSC prior to p5Ocsk has been shown to phosphorylate several Src family PTKs at their carboxyl-terminal , thereby repress- Abbreviations: PTK, protein-tyrosine kinase; GST, glutathione ing their enzymatic function (9-12). Current evidence indi- S-; FSBA, 5'-(p-fluorosulfonylbenzoyl)adenosine; SH, cates that Csk might be a structurally and functionally unique Src homology domain. enzyme, since PTKs related to Csk have not been identified *Present address: Institut fir Medizinische Mikrobiologie und Hy- and because ablation of Csk function in homozygous csk null giene, Technische Universitat Munchen, Munchen, Germany. mice yields an embryonic lethal phenotype (13, 14). tTo whom reprint requests should be addressed at: Bristol-Myers Squibb Pharmaceutical Research Institute, P.O. Box 4000, Prince- ton, NJ 08543-4000. The publication costs ofthis article were defrayed in part by page charge tThe nucleotide sequences reported in this paper have been submit- payment. This article must therefore be hereby marked "advertisement" ted to the GenBank data bank [accession nos. U05210 (murine Ctk) in accordance with 18 U.S.C. ยง1734 solely to indicate this fact. and U05247 (murine Csk)].

2597 Downloaded by guest on September 29, 2021 2598 Biochemistry: Klages et al. Proc. Natl. Acad. Sci. USA 91 (1994) hybridization with 32P-labeled ctk or 1-actin probes. Radio- (Pharmacia), and used to immunize rabbits as previously activity was quantitated with a PhosphorImager using QUANT indicated (23). The resulting antiserum was capable of im- ii software (Molecular Dynamics). munoprecipitating and immunoblotting Ctk but did not de- Production of Ctk and Csk in Sf9 Cells. The Ctk or Csk tectably react with Csk (data not shown). For the in vitro coding sequences were cloned into pBMS-1 (18) and recom- protein kinase reactions using purified enzymes, 2 ug of binant baculoviruses expressing the glutathione S-transfer- 5'-(p-fluorosulfonylbenzoyl)adenosine (FSBA)-treated ase (GST)-Ctk or GST-Csk fusion protein were produced as GST-Lck or 2 pg of a-casein was diluted into kinase buffer described (18). The fusion were purified by glu- (20 mM Mops, pH 7.0/5 mM MnCl2/10 tkM ATP) with 20 ACi tathione affinity chromatography. For excision of the Ctk or of [y-32P]ATP (3000 Ci/mmol; Amersham; 1 Ci = 37 GBq), Csk proteins, purified GST-Ctk or GST-Csk was cleaved and the reactions were initiated by addition of 4 pg of with thrombin and rechromatographed on glutathione col- GST-Ctk or GST-Csk or 2 pg of Ctkc or Cskc (see Results umns (18). Gst-Lck has been described (18). and Discussion). After 10 min at room temperature the Expression in COS Cells. ctk cDNA was subcloned into reactions were stopped with SDS sample buffer. Cyanogen pSV7c (19) and transfected into COS-7 cells (19). The cells bromide peptide analysis of in vivo and in vitro phosphory- were harvested 48 hr later by lysis in a Nonidet P-40- lated p561ck derived from T has been described containing buffer (20) with or without labeling of the cells (24). Phospho amino acid analysis was conducted as de- with [35S]methionine (Amersham) for 3 hr prior to harvest. scribed (24). Biochemical Analyses and Antibody Production. Immuno- precipitations, immune-complex protein kinase assays, and immunoblots have been described (21). For Ctk immunoblots RESULTS AND DISCUSSION from tissues, mouse tissues were harvested and immediately Molecular Cloning ofMurine ctkand cskcDNAs. A two-step frozen in liquid nitrogen. The frozen tissues were pulverized PCR scheme was chosen to enrich for nonreceptor PTK with a mortar and pestle and the cells were lysed in modified genes possibly related to the csk gene. In the first step, PCR RIPA buffer (22). The lysates were cleared by centrifugation, primers were used that corresponded to sequences found in immunoprecipitation of Ctk was conducted by addition of most PTKs, but not in protein-serine/threonine kinases (25). anti-Ctk antiserum, and the presence of Ctk was detected by The second step was performed using PCR primers that Ctk immunoblotting. Anti-Ctk antiserum was produced by would exclude known members ofthe Src family ofPTKs and subcloning the Ctk SH3 domain into the bacterial expression most receptor-type PTKs, as well as many of the known vector pGEX-2T (Pharmacia). The GST-Ctk SH3 fusion nonreceptor PTKs. All primers, however, were predicted to protein was expressed in Escherichia coli, purified on glu- be able to bind to csk-like PITK sequences. DNA from a tathione columns according to the manufacture's instructions mouse whole brain cDNA library was used as template for a I CCGAGTTGAGCCACTGGGTGCTCGACCCGCTGGCAGGGACCCTCCGAGGGTCGCGCCCTMGCAGGTGAG 70 71 CCCTAGATTGTTCGGAGCTTGGACCCCGCGCCTGCTGCAGCTAGGATGCCMCCGCTGGGCCCCTGGG 140 141 ACTCAATGCATGACCAAGTGTGAGAACTCTCGCCCCAAGCCCGGTGAGCTAGCCTTTCGAAAGGGTGACA 210 211 TGGTGACCATCTTrGAGGCCTGTGAGGACAAGAGCTGGTACCGAGCCAAGCACCATGGCAGTGGGCAGGA 280 281 AGGGCTGCTGCGCCGCTGCTCTGCGACACGGGGAGGCCCTCTCCACAGACCCCAAGCTCAGCCTCATG 350 351 CCATGGTTTCATGGCAAGATCTCCGGCCAGGAAGCCATACAGCAGCTGCAGCCACCCGAGGACGGGCTGT 420 421 TCCTTGTGAGWGAATCAGCTCGTCACCCTGGAGACTATGTCTTGTGTGTCAGTTTCGGCCGTGACGTCAT 490 491 CCACTACCGTGTTTTGCATCGAGATGGGCACTCACCATCGATGAGGC GC CCTGATG 560 561 GACATGGTGGAGCACTACACCAAGGACAAGGGGGCCATCTGCACCAAGCTGGTGAAGCCAAGGAGGAAAC 630 631 AGGGCGCAAAGTCTGCAGAGGAGGAGCTCGCCAAGGCTGGCTGGCTACTCGACCTGCAGCATCTGACTCT 700 701 GGGAGCACAGATT lAGAGGGGGAGTTTGGAGCOGTCCTACAGGGTGAGTA CTGGACAGAAGGTGGCT 770 771 GTGAAGAATATCAAGTGTGATGTGACAGCCCAGGCCTTCCTGGATGAGACGGCTGTGATGACGAAGCTGC 840 c 841 AGCACAGGAACCTAGTGCGACTCCTGGGTGTGATCCTGCACCACGGCTTGTACATTGTCATGGAGCACGT 910 ctk 1 MPTR... WAPGTQCMTKCENSRPKPGELAFRKGDMVTILEACEDKSWYRA 47 911 GAGCAAGGGCAACCTGGTGAACTrCCTGCGCACGCGGGGCCGTGCTCTTGTGAGCACCTCTCAGCTTCTG 980 981 CAGTTTGCTCTTCATGTTGCTGAAGGCATGGAATACCTGGAGAGCAAGAAGCTGGTGCACCGGGACCTGG 1050 csk 1 MSAIQAAWPSGTECIAKYNFHGTAEQDLPFCKGDVLTIVAVTKDPNWYKA 50 1051 CTGCTCGGAACATCCTGGTCTCTGAGGACTTGGTGGCCAAGGTCAGTGACTTTGCTTAGCCAAGGCAGA 1120 1121 GCG GGGTGGACTC GCCGGCTGCGTA GTGGACGC CGG 1190 ctk 48 KHHGSGQEGLLAAAALRHGEALSTDPKLSLMPWFHGKISGQEAIQQLQPP 97 1191 TTCTCCAGCAAGTCGGATGTCTGGAGTTTTGGGGTGCTGTTGTGGGAAGTCTTClT ATGGAGAGCCC 1260 1:.111::...1:...111111111111.::1.I II1 1261 CATACCCCAAGATaCCAAAG AGAGGCGT GGGTTACCGCATGGA A 1330 csk 51 KNKV . GREGI IPANYVQKREGVKAGTKLSLMPWFHGKITREQAERLLYPP 99 1331 TGGCTAGGCTCT TGGGTAGCTGCT _ ACCC 1400 1401 T TAGTGGAGAATGGTGTGGGTGTIC 1470 ctk 98 EDGLFLVRESARHPGDYVLCVSFGRDVIHYRVLHRDGHLTIDEAVCFCNL 147 1471 AGGAGGCTGAGGGCTCAGCTCCCACACGGACCAGGA CCCTGATCCCTGAAGCAGAAAGCAAGAGGACA 1540 1541 TAGATGCAGGGTCACAGAGCCAGGAAGAGCTCTGCAGCCCCTGAGACCCCCATGCTGGGACACTCCATA 1610 csk 100 ETGLFLVRESTNYPGDYTLCVSCEGKVEHYRIMYHASKLSIDEEVYFENL 149 1611 AAGAGCATTTGGAAGGATTTTGTAA 1651 ctk 148 MDMVEHYTKDKGAICTKLVKPRRKQGAKSAEEELAKAGWLLDLQHLTLGA 197 csk 150 MQLVEHYTTDADGLCTRLIKPKVMEGTVAAQDEFYRSGWALNMKELKLLQ 199 b 1 ATGTCGGCTATACAGGCCGCCTGGCCATCaGGTACAGAATGTATTCCAAGTACAACTTCCATGGCACTG 70 71 CTGAGCAAGACCTTCCCTTCTGAAAGGAGATGTGCTCACCATCGTGGCTGTCACCAAGGACCCCAACTG 140 247 141 GTACAAAGCCAAAAACAAAGTGGGCCGTGAGGGCATCATCCCAGCCAACTATGTCAGAAGcGTGAGGGT 210 ctk 198 QIGEGEFGAVLQGEYLGQKVAVIKNIKCDVTAQAFLDETAVMTKLQHRNLV 211 GTGAAGGCAGGCACCAAACTCAGCCTTATGCCCTGGTTCCACGGCAAGATCACACGGAGCAGGCCGAG 280 11.1111.1: 1:1 1.11111 111111...1...1 281 GGCTTCTTTACCCACCAGAGACAGGCCTGTTCCTCGTGCGGGAAGCACCAACTACCCTGGAGC CTACAC 350 csk 200 TIGKGEFGDVMLGDYRGNKVAVlCIKNDATAQAFLAEASVMTQLRHSNLV 249 351 ACTGTGTGTGAGCTGTGAGGGCAGGTGGAGCACTACCGCATCATGTATCATGCG.GCMGCTGAGCATT 420 421 GATGAGGAGGTGTACTTTGAGAACCTCATGCAGTGGTGGAGCACTACACCACAGATGCCGATGGACTCT 490 ctk 248 RLLGVILHH. .GLYIVMEHVRKGNLVNFLRTRGRALVSTSQLLQFALHVA 295 491 GCACTCGCCTCATCAAACCAAAGGTCATGGAGGGCACAGTGGCGGCCCAGGATGAGTTCTACCGCAGTGG 560 .11111:.. 11111 1.: 11.11::11.111.:::.. 11.1.1.1. 561 CTGGGCACTGAACATGAAGGAACTGAAGCTGCTACAGACAATAGGGAAGGGGGAGTTTGGAGATGTGATG 630 csk 250 QLLGVIVEEKGGLYIVTEYMAKGSLVDYLRSRGRSVLGGDCLLKFSLDVC 299 631 CTGGGGGATTACCGGGGCAACAAAGTTGCAGTCAAGTGCATCAAGAATGACGCAACTGCCCAGGCCTTCC 700 701 TGGCTGAAGCCTCCGTCATGACGCAACTTCGGCACAGCMCCTCGTCCAGCTGCTGGGTGTGATTGTGGA 770 ctk 296 EGMEYLESKKLVHRDLAARNILVSEDLVAKVSDFGLAKAERKGLDSSRLP 345 771 GGAGAAGGGTGGGCTCTACATCGTCACAGAGTACATGGCCAAGGGGAGTTTGGTGGACTATCTTCGATCA 840 841 CGTGGTCGTTCGGTGCTAGGTGGAGACTGTCTCCTCAAATTCTCATTAGACGTCTTGGAAGCCATGGAGT 910 csk 300 EAMEYLEGNNFVHRDLAARNVLVSEDNVAKVSDFGLTKEASSTQDTGKLP 349 911 ACCTGGAGGGTAACAAlTTlTiTWACCGGGACTTGGCTGCCCGGAATGTGCTGGTGTCTGAAGACAACGT 980 981 GGCCAAAGTCAGTGACTTTGGCCTCACTAAGGAAGCCTCCAGCACTGCAGC 1050 ctk 346 VKWTAPEALKNGRFSSKSDVWSFGVLLWEVFSYGRAPYPKMSLKEVSEAV 395 1051 AMTGGACAGCGCCTGAAGCCTTGAGAGAGAAGAAATTTTCCACCAAGTCTGATGTGTGGAGTTTCGGAA 1120 1121 TCCTTCTCTGGGAAATCTATTCCTTCGGGCGAGTGCCTTACCCAAGAATTCCCCTGAAGGACGTCGTCCC 1190 csk 350 VKWTAPEALREKKFSTKSDVWSFGILLWEIYSFGRVPYPRIPLKDVVPRV 399 1191 TCGGGTGGAAAAGGGCTATAAGATGGACGCTCCGGATGGCTGCCCGCCCGCAGTCTATGATGTTATGAAG 1260 1261 AACTGCTCGCACCTGGATGCrGCCACACGGCCCACGTTTTTGCAGCTTCGGGAACAGCTCGAGCACATCA 1330 1331 AGACCCATGAGCTGCACCTGTGACCTCTGCCTCCGCCAAGGCCATGGGCGCAGGGACAGGGCCTCGGAGA 1400 ctk 396 EKGYRMEPPDGCPGSVHTLMGSCWEAEPARRPPFRKIVEKLG .... RELR 441 1401 TTACAGATCTGGTGCCCTGCTGCTTGCTGGGCCTGAACTTGAACTGAGCCCCAGCCGGCTGGTAGGCCTC 1470 1111:1::11.::I 11.1 .: 1.1: :11: 1471 TTGCCTCTGTACCTGCCTGTCCCTTCTGGCCCCCCAGAGACCCCACCTAGGCCTGGCATCTTCTCTCATG 1540 csk 400 EKGYKMDAPDGCPPAVYEVMKNCWHLDAATRPTFLQLREQLEHIKTHELH 449 1541 GACCCACCTATAW1GCTTGGGGTGCCCACTGAAGGGGCTGAGCAGAGGAGGCCAAGGAACAGGAGAAGTG 1610 1611 CCGTGCAGCTGGCCTCCTCAAGGCCCCACCATTCGCCTTCTTAGAGTTTTATTCCTTTCCTTTTTTGAGA 1680 ctk 442 SVGVSAPAGGQEAEGSAPTRSQDP* 466 1681 TTTTTTTCCGTGMTTTATTTTTTATTATTTTTCAACATAAGGAGAAAGAAAGTACCCAGCAAATGGGCA 1750 1751 TTTTACAAAAA&AAAA A AAAA 1779 csk 450 L* ...... 451 FIG, 1. (a) Nucleotide sequence of the 1651-nt insert of mouse ctk cDNA clone 4. (b) Nucleotide sequence of the 1779-nt insert of a mouse csk cDNA. (c) Alignment of the predicted amino acid sequences (single-letter code) of the mouse ctk and csk gene products. Identical residues are marked with a connecting line, conservative changes with two dots, and similar residues with one dot. The conserved lysine (K) residue at the ATP of protein kinases is presented in bold type. Downloaded by guest on September 29, 2021 Biochemistry: Klages et al. Proc. Natl. Acad. Sci. USA 91 (1994) 2599 PCRs; amplified products were cloned and analyzed by DNA poses, mouse Csk was also produced and purified by the sequencing. Among clones that represented already known same strategy (Fig. 4c). Both GST-Ctk (Fig. 4a, lane 1) and PTK genes, we found one clone possessing novel sequence GST-Csk (lane 5) were found to be capable of autophospho- with the highest homology to csk. rylation as well as phosphorylating a-casein as an exogenous To obtain a complete cDNA for this gene, which was substrate. Both GST-Ctk and GST-Csk were also found to designated ctk (Qsk-type protein tyrosine kinase), the mouse be capable of phosphorylating poly(Glu,Tyr) but did not brain cDNA library that served as source for the PCR phosphorylate purified GST or rabbit muscle enolase (data template was screened with the partial cDNA fragment not shown). Phospho amino acid analysis of the autophos- obtained by PCR. In two screening experiments a total of six phorylated GST-Ctk or GST-Csk as well as the phosphor- independent cDNA clones were identified, containing frag- ments ranging from 1.1 to 2.0 kb. The complete nucleotide a 1 2 3 4 5 sequence ofthe 1.7-kb clone 4 was determined: within a total of 1651 bp, including the poly(A) tail, an open reading frame ctk of 466 codons was identified, extending from a methionine codon at nt 117-119 to a stop codon at nt 1512-1514 (Fig. la). Sequencing the other cDNAs revealed that they encoded the same open reading frame as clone 4 but were either incom- plete at the 5' end or contained larger 5' untranslated se- quences. No evidence for alternatively spliced products was found. Fig. lb shows the complete nucleotide sequence of mouse csk isolated from the same mouse brain cDNA library. Fig. lc shows the alignment ofthe predicted protein products of the mouse csk and ctk genes. Ctk exhibits all hallmarks of PTKs, including Lys221 and surrounding residues, which are highly conserved in protein kinase catalytic regions. The two amino acid stretches that distinguish PTKs from serine/threonine kinases are repre- sented by Ctk sequences DLAARN (aa 311-316) and PVK- WTAPE (aa 345-352) (25). While the 466-aa mouse Ctk possesses only 53% sequence identity to the 451-aa mouse b 2 3 4 5 Csk, both proteins share predicted structural features, in- cluding an SH3 domain (Ctk aa 10-65; Csk aa 14-66), an SH2 domain (Ctk aa 80-154; Csk aa 81-156), and a catalytic actin -4p "O domain or SH1 domain (Ctk aa 191-437; Csk aa 193-440). Iiui.. These homology regions show 33% identity for SH3, 58% for SH2, and 57% for SHi. In addition, both proteins lack amino-terminal sequences, a predicted site for autophosphorylation, and carboxyl-terminal sequences anal- C 1 2 3 4 5 ogous to the Src regulatory tyrosine phosphorylation site. ALg Note that mouse Csk shares about 99%o sequence identity 50 -. * with rat Csk and >90%o sequence identity with chicken and human Csk. 10-. * ctk pS2t Is Expressed Primarily in Brain. Previous analysis of the expression pattern of the 2.4-kb csk mRNA and Csk 2 -w protein indicates that it is most abundant in thymus and spleen (5-9). The 1.9-kb ctk transcript was detected primarily in brain (Fig. 2 a and e), although by dot blot analysis some d expression was detected in spleen and to a lesser extent in liver (Fig. 2c). Quantitation of the dot blot analysis revealed 10. *0 _ 0 0 *acti* that ctk RNA levels were 10- to 20-fold higher in whole brain RNA than in spleen. e 1 Transient expression of ctk in COS cells revealed that the Ctk protein migrated with a molecular mass of -52 kDa (Fig. 3a), which was indistinguishable from the size of Ctk ana- 2. 2.4_ lyzed by in vitro transcription and translation in rabbit 1.9-_- a kb reticulocyte lysates (data not shown). The 52-kDa Ctk was kb also detected in COS cell lysates by immunoblot analysis ctk csk using a Ctk-specific antiserum directed against Ctk aa 2-67 (Fig. 3b). Immunoblot analysis of expression of the 52-kDa FIG. 2. Expression of ctk mRNA in various mouse tissues. (a) Ctk in various mouse tissues showed it to be most abundant Northern blot analysis of whole cell RNA (25 ug per lane) from in brain and at least 10-fold less abundant in spleen (Fig. 3c). various mouse tissues hybridized with 32P-labeled ctk clone 4 cDNA Ctk expression was not detected in the other tissues exam- fragment. The 1.9-kb ctk RNA is indicated by the arrow. Lanes: 1, ined. Thus, the abundance of the Ctk protein in various brain; 2, liver; 3, lung; 4, spleen; 5, kidney. (b) Northern blot analysis mouse tissues was found to correlate with the levels of ctk of the same samples hybridized to a 684-nt mouse P-actin cDNA probe (26). The 1.9-kb P-actin RNA is indicated by the arrow. (c and mRNA derived from the same tissues. d) Dot blots containing the indicated amounts of total RNA from the Ctk Is a P1K That Phosphorylates Src PTKs. To explore same mouse tissues as above hybridized with either the 32P-labeled Ctk function, the Ctk protein was expressed as a GST ctk (c) or (3-actin (d) probe. (e) Northern blots of total RNA (25 pg) chimera in Sf9 insect cells using a baculovirus expression from mouse brain hybridized with the ctk clone 4 probe (panel 1) or system and the enzyme was purified to homogeneity by GST a full-length csk probe (panel 2). The 1.9-kb ctk and 2.4-kb csk affinity chromatography (Fig. 4b). For comparative pur- mRNAs are indicated by arrows. Downloaded by guest on September 29, 2021 2600 Biochemistry: Klages et al. Proc. Natl. Acad. Sci. USA 91 (1994)

a 1 2 3 b 1 2 3 indeed a PTK (data not shown). Cleavage of GST-Ctk and -200 GST-Csk with thrombin to release a portion of the enzyme from GST revealed that Ctkc (Fig. 4a, lane 3) and Cskc (lane 7) were also capable of autophosphorylation as well as phosphorylation of a-casein. In both cases, the cleaved -97 product appeared to be more enzymatically active than the GST chimeras, particularly for the autophosphorylation and a-casein phosphorylation assays. As noted for the chimeras, the reaction products of the cleaved enzymes were exclu- -69 sively phosphotyrosine. .i In parallel experiments, the capacity of GST-Ctk, GST- -: ..:"'. il" Ctkc, and Cskc to phosphorylate purified GST-Lck was 'Mk- "-:: .- ':.':`- .0. Ctk- Csk, -43 evaluated. The Src family member Lck was chosen because ..... "' phosphorylation of Lck at the regulatory Tyr%5 by Csk has been previously documented (10, 11). Forthese experiments, the endogenous protein kinase activity of Lck was inhibited -30 by pretreatment of the purified protein with FSBA, an ATP analog which covalently modifies the enzyme's ATP binding site (12) (Fig. 4a, lane 10). Both Ctk and Csk were capable of i c~ X b- .0 .S phosphorylating the 83-kDa GST-Lck protein (Fig. 4a, lanes CD -i Cn m -* l acid analysis of the Lck c 1 2, 4, 6, and 8). Phospho amino 2 3 4 5 6 7 8 phosphorylated in these assays revealed that phosphoty- -69 rosine was the majorreaction product (Fig. 5 c and d). Similar -Ig results were obtained when FSBA-treated GST-Fyn was Ctk- used as a potential substrate (data not shown). In all cases, -43 Lck was found to be a better substrate (based upon protein FIG. 3. Expression of p52c*. (a and b) Ctk expression in COS concentration) than a-casein. The =60-kDa phosphorylation cells. Lanes: 1, vector alone; 2, lysates of Ctk transfectants immu- product observed in these lanes represents a proteolytic noprecipitated with preimmune serum; 3, lysates ofCtk transfectants product of the GST-Lck cleaved within the GST portion, immunoprecipitated with anti-Ctk antiserum. (a) Cells were labeled since peptide maps of the Ctk- and Csk-phosphorylated with [35S~methionine. (b) Anti-Ctk immunoblot ofanti-Ctk immuno- 83-kDa and 60-kDa protein were indistinguishable (data not precipitates from unlabeled cells. The position ofthe 52-kDa Ctk and shown). immunoglobulin heavy chain are indicated. (c) Anti-Ctk immunoblot cyanogen of anti-Ctk immunoprecipitates from lysates of the indicated mouse Analysis of the GST-Lck reaction products by tissues. Arrows indicate the positions of Ctk and immunoglobulin bromide cleavage demonstrated that the Ctk and Csk en- heavy chain. Lane 1, represents lysis buffer plus anti-Ctk antibody zymes phosphorylated Lck at carboxyl-terminal tyrosine (Ab) alone. Lane 2 represents brain lysate immunoprecipitated with residues within the %4-kDa C3 fagment (Fig. 5), a result formalin-fixed Staphylococcus aureus immunoabsorbant (Pan- compatible with the pattern for phosphorylation of tyrosine sorbin; Calbiochem) alone. Molecular mass markers (prestained; residue 505 (24). In addition, GST-Csk, GST-Ctk, Cskc, and GIBCO; sizes in kilodaltons) are indicated. Ctkc were all capable of phosphorylating a synthetic peptide corresponding to the last 20 aa of Lck (data not shown). ylated a-casein revealed that phosphotyrosine was the only In summary, the results indicate that we have isolated a detectable reaction product, thereby establishing that Ctk is cDNA for a PTK that represents the second example of an

a Gst- Ctk Ctkc GstICsk CskC Control

1 2 3 4 5 6 7 8 9 10 -200 b w

_97 1 2 3 12 3 Gst-Lckip-| tt: Gst- Ct k /Cskw- -69 -200 1 -200

Ctkc/CskII. -97 -97 -43 -6S -43 .3.0 caseinim ..:-- .-30 -30

FIG. 4. Comparison of purified Ctk and Csk functions in vitro. (a) Purified GST-Ctk Qanes 1 and 2), Ctkc (lanes 3 and 4), GST-Csk (lanes 5 and 6), and CskC (lanes 7 and 8) were evaluated for their ability to phosphorylate a-casein (lanes 1, 3, 5, and 7) or FSBA-taed GST-Lck Oanes 2, 4, 6, and 8). Phosphorylation of a-casein and FSBA-treated GST-Lck in the absence of added protein kine is shown in lanes 9 and 10, respectively. (b) Coomassic blue-stained SDS/polyacrylamide gel of GST-Ctk baculovirus-nfected 519 lysates (lane 1), purified GST-Ctk (lane 2), and purified GST-Ctk following cleavage with thombin to release a portion of the Ctk enzyme from the GST protein. (c) Coomassie blue-stained SbS/polyacrylamide gel of GST-Csk baculovirus-infected Sf9 lysates (lane 1), purified GST-Csk (lane 2), and purified GST-Csk following cleavage with thrombin to release a portion of the Csk enzyme from the GST protein. Positions of GST-Lck, GST-Ctk, GST-Csk, Ctkc, Cskc, R-casein, and molecular mass markers (prestained; GIBCO) are indicated. Position of a proteolytic product ofGST-Lck representing the majority of the Lck protein is indicated by the asterisks. Downloaded by guest on September 29, 2021 ..-30 Biochemistry: Klages et al. Proc. Natl. Acad. Sci. USA 91 (1994) 2601

a 2 1. Cooper, J. A. (1989) in Peptides andProtein Phosphatases, ed. 43- Kemp, B. (CRC, Boca Raton, FL), pp. 85-113. 2. Bolen, J. B., Rowley, R. B., Spana, C. & Tsygankov, A. Y. cl 30- (1992) FASEB J. 6, 3403-3409. 21- S y394 y505 3. Hunter, T. (1987) Cell 49, 1-4. 14- N C 4. Cantley, L. C., Auger, K. R., Carpenter, C., Duckworth, B., 6- Ipi tI------.--- -4 2 C------Granziani, A., Kapeller, R. & Soltoff, S. (1991) Cell 64, ci C2 C3 281-302. C3-.- w 5. Nada, S., Okada, M., MacAuley, A., Cooper, J. A. & Naka- gawa, H. (1991) Nature (London) 351, 69-72. 6. Sabe, H., Knudsen, B., Okada, M., Nada, S., Nakagawa, H. 1 2 3 & Hanafusa, H. (1992) Proc. Natl. Acad. Sci. USA 89, 2190- 43- c d 2194. 7. Partanen, J., Armstrong, E., Bergman, M., Makela, T. P., Cl-_ 30- _ ..S. t. Hirvonen, H., Huebner, K. & Alitalo, K. (1991) Oncogene 6, 21- S 2013-2018.

14- y 8. Brauninger, A., Holtrich, U., Strebhardt, K. & Rubsamen- 6- Weigmann, H. (1992) Gene 110, 205-211. C3-_b , 9. Okada, M., Nada, S., Yamanashi, Y., Yamamoto, T. & Nak- agawa, H. (1991) J. Biol. Chem. 266, 24249-24252. FIG. 5. Phosphopeptide and phospho amino acid analysis of Csk 10. Bergman, M., Mustelin, T., Oetken, C., Partanen, J., Flint, and Ctk reaction products. (a and b) SDS/polyacrylamide gel analysis N. A., Amrein, K. E., Autero, M., Burn, P. & Alitalo, K. of 32P-labeled Lck cyanogen bromide peptides. (a) Lck from 32Pi- (1992) EMBO J. 11, 2919-2924. labeled human T lymphocytes (lane 1) or Lck autophosphorylated in 11. Chow, L. M. L., Fournel, M., Davidson, D. & Veillette, A. immune-complex kinase assays (lane 2), demonstrating the fraction- (1993) Nature (London) 365, 156-160. ation profile of the major cyanogen bromide peptides. (b) Lck from 12. Okada, M. & Nakagawa, H. (1989) J. Biol. Chem. 264, 20886- 32P,-labeled T cells (lane 1) or FSBA-treated GST-Lck phosphorylated 20893. in vitro by either GST-Csk (lane 2) or GST-Ctk (lane 3). Positions of 13. Imamoto, A. & Soriano, P. (1993) Cell 73, 1117-1124. prestained molecular mass markers and phosphopeptides are indicated. 14. Nada, S., Yagi, T., Takeda, H., Tokunaga, T., Nakagawa, H., A schematic representation ofthe cyanogen bromide cleavage sites C1, Ikawa, Y., Okada, M. & Aizawa, S. (1993) Cell 73, 1125-1135. C2, andC3 ofLck are shown at right. The C1 peptide contains primarily 15. Innis, M. A., Gelfand, D. H., Sninsky, J. J. & White, T. J., serine phosphorylation sites, while C2 contains the Tyr394 autophos- eds. (1990) PCR Protocols: A Guide to Methods and Applica- phorylation site and C3 contains the Tyr"5 Csk phosphorylation site. tions (Academic, New York). Arrowheads indicate the predicted site of cyanogen bromide cleavage. 16. Chirgwin, J. M., Przbyla, A. E., MacDonald, R. J. & Rutter, (c and d) Phospho amino acid analysis of FSBA-treated GST-Lck W. J. (1979) Biochemistry 18, 5294-5299. phosphorylated in vitro by either GST-Csk (c) or GST-Ctk (d). Posi- 17. Thomas, P. S. (1980) Proc. Natl. Acad. Sci. USA 77, 5201- tions ofphospho amino acid standards (S, phosphoserine; T, phospho- 5205. threonine; Y, phosphotyrosine) are indicated. 18. Spana, C., O'Rourke, E., Bolen, J. B. & Fargnoli, J. (1993) Protein Expression Purification 4, 390-397. 19. Adam, D., Klages, S., Bishop, P., Mahajan, S., Escobedo, J. enzyme capable of phosphorylating members of the Src & Bolen, J. B. (1993) J. Biol. Chem. 268, 19882-19888. family of PTKs at their regulatory sites. This enzyme, Ctk, 20. Burkhardt, A. L. & Bolen, J. B. (1993) Curr. Protocols Immu- while distinguishable from Csk by sequence, mRNA size, and nol. Suppl. 7, Unit 11.4. tissue distribution, is similar to Csk from a predicted struc- 21. Veillette, A., Bookman, M. A., Horak, E. M. & Bolen, J. B. tural and demonstrated functional standpoint. In addition, (1988) Cell 55, 301-308. molecular cloning and sequencing ofthe genomic counterpart 22. DeSeau, V., Rosen, N. & Bolen, J. B. (1987) J. Cell. Biochem. 35, 113-128. of the mouse ctk cDNA have established that ctk has an 23. Burkhardt, A. L., Brunswick, M., Bolen, J. B. & Mond, J. J. exon-intron organization very similar to that of csk (P. M. (1991) Proc. Natl. Acad. Sci. USA 88, 7410-7414. Brinkley and R.C.P., unpublished results). Therefore, our 24. Veillette, A., Bolen, J. B. & Bookman, M. A. (1989) Mol. Cell. results demonstrate that Ctk represents a PTK that, together Biol. 9, 4441-4446. with Csk, defines the family of Csk-type enzymes. 25. Hanks, S. K., Quinn, A. M. & Hunter, T. (1988) Science 241, 42-52. We thank Ed O'Rourke and Hilary Gray for isolation and produc- 26. Tokunaga, K., Taniguchi, H., Yoda, K., Shimizu, M. & tion of the recombinant baculoviruses used in this work. Sakiyama, S. (1986) Nucleic Acids Res. 14, 2829. Downloaded by guest on September 29, 2021