THE JOURNAL OF BIOLOGICAL CHEMISTRY C 1991 by The American Society for Biochemistry and Molecular Biology, Inc

Vol . 266, No. 24, Issue of August 25, pp. 16073-16077.1991 Printed in U. S. A.

TIK, a Novel Serine/Threonine Kinase, Is Recognized by Antibodies Directed against Phosphotyrosine*

(Received for publication, March 28, 1991)

Pamela L. Icely$, Philippe Grospll, John J. M. Bergeronll , Alain Devaultp, Daniel E. H. Afar$, and John C. Bell$** From the $Departments of Medicine and Biochemistry, University of Ottawa, 451 Smyth Road, Ottawa, Ontario KlH 8M5, Canada, the Departments of §Biochemistry and IIAnatomy, McGill Uniuersity, Montreal, Quebec H3G IY6, Canada

We have isolated cDNAs encoding kinases from a murine pre-B cell line by screening a hgtl l cDNA expression library with anti-phosphotyrosine antibod- ies. One cDNA was identified to encode the previously isolated tyrosine kinase c-lyn. Among the remaining clones, we have characterized a cDNA encoding a novel kinase which we have designated TIK. Sequence analysis of this cDNA indicates that the TIK enzyme lacks the features thought to be conserved among pro- tein tyrosine kinases. Although isolated on the basis of its reactivity with the anti-phosphotyrosine antibody, the TIK enzyme was found to have only serine and threonine kinase activity. The amino-terminal portion of the TIK protein contains a cdc2 phosphorylation consensus sequence. Three mRNA transcripts derived from the TIK gene are detected in a variety of adult murine tissues.

Molecules comprising cellular signal transduction networks provide a link from extracellular stimuli to the internal envi- ronment of the cell. Currently, attempts are being made to dissect the pathways that transduce signals controlling cel- lular growth and development. Some protein tyrosine kinases have been identified as growth factor receptors, clearly de- picting these enzymes as mediators of cell proliferation and differentiation signals (1). Recent identification of serine/ threonine kinases as downstream effectors of specific tyrosine kinases indicates that cellular signal transduction requires the coordinated action of a variety of kinases (2).

It has become apparent that the regulated expression of specific tyrosine kinases during hematopoiesis is crucial to normal hematopoietic development. Mutations at the white locus of the mouse, which has been identified to encode the tyrosine kinase c-kit (3), lead to developmental defects in- cluding severe macrocytic anemia and mast cell deficiency. In addition to playing a role in the transduction of signals controlling cell growth and development, some src-related tyrosine kinases have specialized functions in fully differen- tiated hematopoietic cells (4).

* This work was supported by a grant from the Medical Research Council of Canada (to J. C. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisernent" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession numberfs) M65029.

ll Supported by a Scientist Award from the Fonds de Recherche en Santii du Quebec.

** A Medical Research Council scholar. To whom correspondence should be addressed. Tel.: 613-787-6698; Fax: 613-738-3191.

Enzymatically active tyrosine kinases undergo autophos- phorylation, allowing their detection by antibodies to phos- photyrosine. Such antibodies have been successfully used in a variety of systems to screen cDNA expression libraries for cDNAs encoding functional tyrosine kinases (5-7). Using this method, we have isolated a number of cDNA clones from a pre-B cell expression library. One cDNA was found to encode a kinase which appeared to be more closely related to serine/ threonine kinases than tyrosine kinases. We present evidence here that although this novel kinase can be recognized by the anti-phosphotyrosine antibody, it is in fact a serinelthreonine kinase.

MATERIALS AND METHODS

Isolation of cDNA Clones

cDNAs encoding anti-phosphotyrosine immunoreactive proteins were isolated from a hgt l l cDNA expression library generated from poly(A)+-selected RNA from the 70Z/3 cell line. 5 X lo5 plaques were screened by infecting the bacterial strain Y1090 with the bacterio- phage and incubating at 42 "C for 4-6 h, overlaying with filters soaked in 10 mM isopropyl-(3-D-thiogalactopyranoside, and incubating for an additional 6 h a t 37 "C. Filters were then treated with polyclonal murine anti-phosphotyrosine antibody and anti-mouse antibody cou- pled to alkaline phophatase. Positive clones were visualized through reaction with Nitro Blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate. These clones were rescreened twice to homogeneity.

Positive clones were cross-hybridized to identify distinct clones by the following method: 1 p1 of phage in solution a t 10 pfu was spotted onto a plated bacterial lawn of Y1090, allowed to adsorb for 20 min at 22 "C, and incubated for 8-12 h a t 37 "C. Nitrocellulose filters were overlayed for 4 min, denatured in 1.5 M NaCl, 0.5 M NaOH for 2 min, neutralized in 1.5 M NaC1, 0.5 M Tris-C1, pH 8, for 5 min, and rinsed in 0.2 M Tris-C1, pH 7.5,2 X SSC for 30 s. Filters were baked a t 80 "C under vacuum for 2 h. Filters were probed with DNA probes generated by random primer extension labeling of polymerase chain reaction- amplified cDNA inserts. Prehybridizations were carried out with 6 X SSC, 5 X Denhardt's solution, 0.5% SDS,' and denatured salmon sperm DNA at 0.1 mg/ml a t 65 "C for 6 h. Hybridizations were performed using radioactively labeled probe a t 1-4 X lo5 cpm/ml for 12 h. Filters were washed in 2 X SSC at 22 "C for 5 min and once in 0.1 X SSC, 0.1% SDS at 22 "C for 15 min, then autoradiographed by exposure to Kodak XAR-5 x-ray film.

Sequencing of cDNA Clones

The cDNA inserts were cloned into the EcoRI site of the bacteri- ophage M13 (Pharmacia LKB Biotechnology Inc.), and single- stranded sequence analysis was carried out using the Sanger dideoxy chain termination method (25). Predicted amino acid sequences were compared with the proteins of the National Biomedical Research Foundation protein data bank using the RUNFASTP program.

The abbreviations used are: SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; kb, kilobase(s); Mops, 4-morpho- linepropanesulfonic acid; HEPES, N-2-hydroxyethylpiperazine-N'- 2-ethanesulfonic acid.

16073

16074 Characterization of the TIK Kinase RNA Isolation and Northern Blot Analysis

Total RNA was prepared from cells or tissues by the method of Auffray and Rougeon (26). Poly(A)' selection was carried out by passage of total RNA over oligo(dT)-cellulose, following the method of Jacobson (27). Aliquots of 4 pg of poly(A)+ RNA were electropho- resed in 1% agarose gels containing 2.2 M formaldehyde, 20 mM Mops (pH 6.8), 1 mM EDTA, and 5 mM sodium acetate, and the electro- phoretically separated RNAs were transferred to Hybond-N mem- brane. Filters were baked for 2 h a t 80 "C and treated with ultraviolet light for 5 min. Prehybridization was carried out 12-24 h in 5 X SSPE, 50% formamide, 2 mM NaPi, pH 6.8,5 X Denhardt's solution, 5% dextran sulfate, 0.5% SDS, and 250 pg/ml denatured nonhomol- ogous DNA, at 42 "C. Hybridizations were performed using a random- primed '"P-labeled DNA insert a t 1-2 X lo6 cpm/ml a t 42 "C for 12- 24 h. Filters were washed to a final stringency of 0.1 X SSC, 0.1% SDS at 22 "C, and exposed to Kodak XAR-5 x-ray film.

Bacterial Expression of the TIK cDNA The entire coding region of the TIK cDNA from the BamHI site

(position 116) 21 base pairs upstream of the putative initiating methionine residue to position 1849 (146 base pairs 3' to the stop codon) was subcloned into the BamHI site of the pETllc expression vector (28,29). Escherichia coli pLysS containing this construct was typically induced to express the T7 gene 10 product-TIK fusion protein by growing the bacteria to an ODm of 0.6 in LB medium containing 50 pg/ml ampicillin, and adding 0.4 mM isopropyl-8-D- thiogalactopyranoside to the bacterial medium and growing for 2 h.

Protein and Phosphoamino Acid Analysis I n Vitro Labeling Experiments-E. coli pLysS bacteria containing

the TIK sense or antisense pETllc construct were induced (as described above) to express the T7 gene 10 product-TIK fusion protein and lysed by freeze-thawing. Lysates were cleared by centrif- ugation, and the supernatants were immunoprecipitated with either PY20 (from ICN Biochemicals), IgG2bk (from Upstate Biotechnol- ogy, Inc.), MAlG2 (generous gift of Dr. A. Raymond Frackelton, Jr., Brown University), or a polyclonal rabbit anti-phosphotyrosine an- tibody, in 10 mM Tris (pH 7.5), 150 mM NaC1, 5 mM EDTA, 1% Triton X-100, 2 mM NaF, 2 mM sodium pyrophosphate, 500 p M ammonium vanadate, and 200 pg of phenylmethylsulfonyl fluoride per ml. The immunoprecipitates were assayed for kinase activity in 20 mM HEPES, pH 7.1, and 10 mM MnC12 with 0.1 mCi/ml [r-"P] ATP for 30 min at 22 "C. The reaction products were resolved by 10% SDS-polyacrylamide gel electrophoresis, and the dried gels were exposed to Kodak XAR-5 film. Phosphoproteins were electroeluted for phosphoamino acid analysis by the method of Edwards et al. (30).

In Vivo Labeling Experiments-Bacteria harboring the TIK sense or antisense expression construct were grown to an ODW, spun down, and resuspended in 50 mM Tris (pH 7.5), 100 mM NaC1, 10 mM MgCI2, and 10 mM MnC12. Either ["S]methionine or [32PJorthophos- phate were added to 10 pCi/ml or 2 mCi/ml, respectively. The bacteria were then induced (as described above) and incubated a t 37 "C with shaking for 1-2 h, lysed, immunoprecipitated, and the products ana- lyzed as described above.

Kinase Renaturation Assay-This procedure was carried out essen- tially as described in Ferrell and Martin (18). Whole cell lysates from induced bacteria were resolved by 10% SDS-PAGE and transferred

47 15 tik 8 9 30 40 ---._., 1

" 18s-

FIG. 1. Northern blot analysis showing mRNA sizes of the seven distinct cDNAs isolated from the 702/3 cell line. 5 pg of poly(A)+-selected RNA was used for each lane of the gel and hybrid- ized to a cDNA probe generated for each cDNA. The 18 and 28 S rRNAs are 1869 and 4712 nucleotides, respectively.

I tD cu -. rc

r C

CCI I

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FIG. 2. Northern blot analysis of TIK mRNA in a variety of adult mouse tissues. Each lane contains approximately 5 pg of poly(A)+-selected RNA isolated from each mouse tissue. The blot was hybridized to a TIK cDNA and murine 8-actin probe. The 18 and 28 S rRNAs are 1869 and 4712 nucleotides, respectively.

to Immobilon P membranes. Blotted proteins were denatured in 6 M guanidinium chloride, 50 mM Tris, 50 mM dithiothreitol, and 2 mM EDTA, and then renatured overnight at 4 "C in 100 mM NaCI, 50 mM Tris, 2 mM dithiothreitol, 2 mM EDTA, and 0.1% (w/v) Nonidet P-40 (ph 7.5). Blots were then blocked with 5% (w/v) albumin in 30 mM Tris (pH 7.5) a t room temperature for 1 h. The kinase assay was performed by incubating blocked blots in 30 mM Tris, 10 mM MgC12, and 10 mM MnCI2 (pH 7.5), with 50 pCi/ml [y3'P]ATP at room temperature for 30 min. Blots were washed twice with 30 mM Tris (pH 7.5), followed by 30 mM Tris (pH 7.5) and 0.05% Nonidet P-40 for 10 minutes a t room temperature prior to autoradiography.

RESULTS

Isolation of cDNA Clones-We have used an antibody to phosphotyrosine to screen a X g t l l cDNA expression library prepared with mRNA isolated from the pre-B cell line 70Z/3 (8). From the screening of 5 X lo5 recombinant plaques we identified eight distinct clones encoding fusion proteins re- acting with a polyclonal anti-phosphotyrosine antibody. Through cDNA sequence analysis, one cDNA was identified as the tyrosine kinase c-lyn (9), verifying the efficacy of this screening procedure. The mRNA sizes for the remaining seven clones were determined by Northern blot analysis using poly(A)+-selected RNA from the 70Z/3 line (Fig. 1). In agree- ment with their designation as distinct gene products, these cDNAs hybridize to mRNAs of varying abundance and tran- script size. It has been shown previously that the tyrosine kinase c-ab1 gene is expressed in 70Z/3 cells (8). None of our clones, however, were found to hybridize with a c-ab1 probe.

Characterization of the TIK Kinase-One novel kinase

Characterization of the TIK Kinase 16075

TI[ K W . L

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FIG. 3. The nucleotide and predicted amino acid sequence of the TIK cDNA. The cdc2 phosphorylation consensus sequence is boxed, and the residues making up the catalytic domain are under- lined.

DLKPEN S\T K W A S

DLRAAN Y KINASE

I VY

TLQYMSPE TI( KWASE

TXXYXAPE S\l KINASE

PIKWTAPE Y KINASE

I I L

FIG. 4. A schematic representation of the TIK kinase. Serine/threonine and tyrosine kinase subdomain consensus se- quences are indicated by Roman numerals. X, any amino acid. Boxed amino acids represent conserved residues.

A B

1 2 1 2

97 - 68 -

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FIG. 5. In vivo labeling of the TIK kinase. Lysates of E. coli pLysS expressing the TIK fusion protein in either the sense ( A ) or antisense ( B ) orientation in the presence of ["S]methionine. Lane I, ["S]methionine added at the time of induction and bacteria harvested after 1.5 h; lane 2, [:%]methionine added 0.5 h postinduction and bacteria harvested after 1 h.

which we designated TIK, for anti-phosphotyrosine immu- noreactive kinase, was selected for further study. A 2.1-kb TIK cDNA hybridizes to three mRNAs of 6,4, and 2.5 kb in the 70Z/3 line (Fig. 1). Transcripts hybridizing to the TIK cDNA are also detected in murine heart, lung, brain, kidney, testes, thymus, and bone marrow (Fig. 2). There are tissue- specific differences in the ratios of the three TIK transcripts; for example in both lung and heart tissue the 4-kb mRNA is predominant over the 6- and 2.5-kb mRNAs (Fig. 2). The 2.5- kb mRNA is expressed a t high levels in testes tissue, suggest- ing that TIK may play a role in spermatogenesis. High strin- gency hybridization of the complete TIK cDNA to 70Z/3 DNA digested with the restriction endonucleases HindIII, BamHI and KpnI produced a very simple pattern of bands, indicating that the three mRNAs originate from a single gene (data not shown).

The nucleotide sequence of the TIK cDNA is shown in Fig. 3. The sequence contains an open reading frame of 517 amino acids ending a t position 1701. The putative initiating methi- onine residue at nucleotide position 147 is preceded by one

16076

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Characterization of the TIK Kinase

D 1 2

F 1 2

PS PT

PY

PS

PT

PY

FIG. 6. A, in uitro kinase reactions of the TIK fusion protein in bacterial lysates. Lysates of E. coli pLysS expressing the TIK fusion protein in either the sense (lane 2 ) or antisense (lane I ) orientation were immunoprecipitated with an anti-phosphotyrosine antibody and assayed for kinase activity in the presence of [r-:"P]ATP prior to SDS-polyacrylamide gel electrophoresis. B, phosphoamino acid analysis of electroeluted products from panel A. The positions of the phosphoamino acid standards are indicated as follows: pS , phospho- serine; pT, phosphothreonine; and pY, phosphotyrosine. C, in uiuo labeling of the TIK fusion protein. E. coli pLysS expressing the TIK fusion protein in either the sense (lane I ) or antisense (lane 2 ) orientation were grown in the presence of [R2P]orthophosphate. Ly- sates from these bacteria were resolved on SDS-polyacrylamide gel electrophoresis and autoradiographed. D, phosphoamino acid analysis of electroeluted products from panel C. E , kinase renaturation assay of the TIK kinase. Lysates of E. coli pLysS expressing the TIK fusion protein in either the sense (lane I ) or antisense (lane 2 ) orientation

termination codon, and the sequence surrounding this ATG site conforms to the consensus predicited by Kozak's rules for ribosome binding (10). The TIK protein predicted from the cDNA sequence would have a molecular mass of 58,573 Da, with an isoelectric point of 8.38.

The presumed catalytic region of the TIK enzyme is con- tained within amino acid residues 252-505. When compared with the NBRF-PIR(r) data base using the Lipman and Pearson algorithm (ll), TIK was found to share a high degree of amino acid sequence identity with serinelthreonine kinases. The highest degree of identity was found with the cdc2 kinase (27% identity in a 185-amino acid overlap). In general, the catalytic domain of protein kinases contains conserved regions designated by Hanks et al. (12) as subdo- mains I-XI. As the TIK kinase was cloned by virtue of its immunoreactivity with the anti-phosphotyrosine antibody, we expected its conserved subdomains would most closely resem- ble those of the tyrosine kinase family. The primary sequences of subdomains VI and VI11 are thought to be indicative of the hydroxyamino acid specificity of the kinase. As illustrated in Fig. 4, these regions in TIK match more closely the serine/ threonine rather than the tyrosine kinase consensus sequence.

Other features of the TIK kinase which may be of signifi- cance lie amino-terminal to the catalytic domain. This region of the predicted TIK polypeptide (residues 1-252) shares little homology with previously identified serinelthreonine or ty- rosine kinases. The primary sequence does not provide a clear indication of the subcellular localization of the TIK enzyme. The kinase has no apparent transmembrane domain, nor does it have a myristylation site a t residue 2, which is typically found in tyrosine kinases targeted to membrane surfaces (13). The TIK protein does not have an SH-2 domain (14), which is found in non-receptor-type tyrosine kinases and other enzymes of the signal transduction network.

The kinase recognition sequence for cdc2 phosphorylation, Ser/Thr-Pro-Xaa-Arg/Lys (15), is found at residues 157-161 of the TIK protein (Fig. 3). This consensus sequence is followed by several serine and threonine residues, in particular two clusters of serine residues.

Kinase Activity of TIK-The observation that the catalytic domain of the TIK kinase resembled more closely the catalytic domains of serinelthreonine kinases than those of tyrosine kinases prompted us to investigate the biochemical activity of this enzyme. The TIK kinase was expressed in bacteria as a fusion protein, using a cDNA construct in which the coding region of the T7 gene 10 product is fused in frame with the 5' end of the entire TIK coding region. I n vitro transcription and translation of this TIK fusion protein produced a poly- peptide of approximately 67 kDa (data not shown). The expected molecular mass of the TIK fusion protein was ap- proximately 60 kDa, so it appeared to be migrating anomo- lously in the SDS-PAGE system. Other phosphoproteins also migrate to unexpected apparent molecular masses in SDS- PAGE systems, for example the E1A protein of adenovirus (16) and the NS protein of vesicular stomatitus virus (17).

When intact bacteria expressing the TIK fusion protein were metabolically labeled with ["'S]methionine, a 67-kDa polypeptide was produced that can be immunoprecipitated with antibodies to phosphotyrosine (Fig. 5). Bacteria express- ing an identical TIK antisense construct did not produce this

were resolved by SDS-PAGE and transferred to an Immobilon P membrane. The blotted proteins were denatured and renatured as described under "Materials and Methods" and assayed for kinase activity in the presence of [Y-~'P]ATP. The blot was then washed and autoradiographed. F, phosphoamino acid analysis of the protein blotted onto Immobilon P shown in panel E.

Characterization of the TIK Kinase 16077

polypeptide, confirming the identity of this 67-kDa polypep- tide with the TIK fusion protein produced by in vitro tran- scription and translation. The immunoprecipitated 67-kDa protein was found to have an associated kinase activity when incubated with [-y-32P]ATP in an in vitro kinase assay (Fig. 6A). Phosphoamino acid analysis shows that this protein contains phosphoserine and phosphothreonine, but not phos- photyrosine (Fig. 6B). In an experiment in which bacteria expressing the TIK fusion protein were labeled in vivo with [32P]orthophosphate, a labeled 67-kDa protein was immuno- precipitated with anti-phosphotyrosine antibodies (see Fig. 6C). Phosphoamino acid analysis of this 67-kDa band showed phosphoserine and phosphothreonine, but no phosphotyro- sine (see Fig. 6D).

Although these experiments strongly suggest that the 67- kDa TIK fusion protein has intrinsic serinelthreonine kinase activity, it remained formally possible that this activity is attributable to an associated bacterial kinase. To rule out this possibility, we performed a kinase renaturation assay. In this procedure, lysates from bacteria expressing the TIK fusion protein were separated by gel electrophoresis, blotted to an ImmobiIon P membrane, induced to renature as described by Ferrell and Martin (18), and incubated with [y3'P]ATP. The renatured 67-kDa TIK fusion protein incorporates radioac- tively labeled phosphate (Fig. 6E). Lysates from bacteria expressing the TIK antisense construct did not show this activity. Phosphoamino acid analysis of the renatured TIK kinase showed that it contains phosphoserine and phospho- threonine (see Fig. 6F), confirming that the TIK protein has intrinsic serinelthreonine kinase activity.

DISCUSSION

We have cloned and characterized a novel kinase, TIK, from the pre-B cell line 702/3. TIK has intrinsic serine and threonine kinase activity, both in in vitro kinase assays and in vivo in bacteria. Under no conditions was this kinase found to have tyrosine phosphorylating ability. This finding is in agreement with our observation that the catalytic domain of TIK contains residues thought to be conserved among serine/ threonine kinases, but not tyrosine kinases (12) (Fig. 4). When compared with the proteins of the NBRF data bank, the TIK kinase was found to share the highest degree of identity with the serinelthreonine kinase cdcP Members of the cdc2 protein kinase family typically contain a short sequence of strong homology near subdomain XI of their catalytic domains. Although the region of shared identity between the TIK and cdc2 kinases is found in the catalytic domain, it does not include the sequences surrounding subdomain XI.

The TIK cDNA hybridizes to three mRNA transcripts in all mouse tissues analyzed (Fig. 2). Other single-copy kinase genes have been shown to give rise to multiple mRNAs through alternative splicing of a single mRNA species or differential promoter usage (19-24). These additional tran- scripts encode proteins differing in specific domains. While the size difference of the TIK transcripts could represent varying untranslated regions, it is possible that these mRNAs give rise to three isoforms of this enzyme.

Despite its lack of phosphotyrosyl residues, the TIK protein can be detected and immunoprecipitated with three different monoclonal anti-phosphotyrosine antibodies and a polyclonal anti-phosphotyrosine antibody. A possible explanation for this apparent paradox is that these antibodies are cross-

reacting with phosphoserine or phosphothreonine. However, detection of the 67-kDa TIK fusion protein band in anti- phosphotyrosine immunoblots of lysates from TIK expressing cells was blocked by competition with 5 mM phosphotyrosine, but not with phosphoserine or phosphothreonine (data not shown). This observation suggests that an epitope resembling phosphotyrosine may be encoded by the TIK primary se- quence. If indeed such an epitope does exist it may play a role in the regulation of the biochemical and biological properties of the TIK kinase. The TIK epitope detected by the anti- phosphotyrosine antibodies may represent a binding site for molecules which specifically recognize phosphotyrosyl resi- dues and could therefore allow the TIK kinase to gain access to signal transduction molecules which normally communi- cate via phosphotyr0sine:protein interactions. Experiments designed to test this hypothesis are currently underway.

Use of the anti-phosphotyrosine antibody has been consid- ered a specific screen for functional tyrosine kinases (5-7). Our observation that the TIK kinase reacts with such anti- bodies but does not possess tyrosine phosphorylating ability indicates that this screening procedure also unexpectedly detects novel serinelthreonine kinases.

Acknowledgments-We thank Dr. D. A. Gray and Dr. W. M. McBurney for constructive criticism of the work presented here and R. Marius and N. Ooman for expert technical assistance. We also thank B. Howell, E. Douville, P. Duncan, and T. Marshall for critical reading of this manuscript.

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