©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of Rlk, a Novel Protein Tyrosine Kinase with Predominant Expression in the T Cell Lineage (*)

(Received for publication, June 15, 1994; and in revised form, November 8, 1994)

Qile Hu (1)(§) David Davidson(§) (2)(¶) Pamela L. Schwartzberg (4)(**) Francesca Macchiarini (5) Michael J. Lenardo (4) Jeffrey A. Bluestone (3) Louis A. Matis (§§)

From the  (1)Immunobiology Program, Alexion Pharmaceuticals, Inc., New Haven, Connecticut 06511, the Biological Response Modifiers Program, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, Maryland 21702, the Departments of (2)Endocrinology and (3)Pathology, University of Chicago, Chicago, Illinois 60637, the (4)Laboratory of Immunology, NIAID, National Institutes of Health, Bethesda, Maryland 20098, and the (5)Biological Carcinogenesis and Development Program, Program Resources, Inc./DynCorp, Frederick, Maryland 21702

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The control of phosphorylation by protein tyrosine kinases represents an important regulatory mechanism in T cell growth, function, and differentiation. We have identified a 62-kDa murine protein tyrosine kinase predominantly expressed within the T cell lineage, which we have termed Rlk (for Resting lymphocyte kinase). rlk mRNA was found to be expressed in the fetal thymus as early as day 13 of embryonic development as well as in adult thymus and mature resting peripheral T cells. The sequence of rlk showed that it is most closely related to the subfamily of cytoplasmic tyrosine kinases that includes the Btk, Itk, and Tec proteins. However, Rlk differs from these kinases by virtue of its unique amino-terminal domain, which lacks a region of pleckstrin homology common to the other members of this protein subfamily. Examination of rlk abundance within different T cell subpopulations revealed preferential expression in Th1 relative to Th2 T cell clones, suggesting a possible role in signal transduction pathways that selectively regulate cytokine production in mature CD4 T cell subsets. Rlk thus represents a novel cytoplasmic tyrosine kinase with potential functions in intrathymic T cell development and mature T cell signaling.


INTRODUCTION

The regulation of protein tyrosine phosphorylation is a critical component of signal transduction during cell growth and differentiation. This is well illustrated in lymphocytes, where the initiation of signaling through antigen receptors is associated with the rapid tyrosine phosphorylation of multiple intracellular substrates (1, 2, 3) . There is substantial biochemical and genetic evidence for the participation of protein tyrosine kinases in mature T cell activation as well as in T cell development. For example, two Src-related enzymes, FynT and Lck, have key roles in T cell receptor-mediated signaling (4, 5, 6, 7, 8) . A third cytoplasmic protein tyrosine kinase with expression restricted to the T cell lineage, ZAP-70, rapidly associates via its two SH2 domains with tyrosine-phosphorylated antigen recognition motifs of the CD3 complex following T cell receptor stimulation(4, 6) . Members of the Csk kinase family negatively regulate the enzymatic activity of the Src kinases via the specific phosphorylation of a carboxyl-terminal regulatory tyrosine(9) . Overexpression of Csk in antigen-specific T cell lines inhibits responsiveness following antigen stimulation(10) .

The most recently identified family of nonreceptor tyrosine kinases includes the interleukin-2-inducible kinase Itk, the Bruton's agammaglobulinemia tyrosine kinase Btk, and the Tec kinase(11, 12, 13, 14, 15, 16) . Although sharing structural features with the Src kinases, the proteins of this family lack the negative regulatory carboxyl-terminal tyrosine as well as the amino-terminal consensus sequence for myristylation that is characteristic of Src family members. Both Itk and Btk also display restricted patterns of expression, Itk predominantly in the T cell lineage (11, 12) and Btk in myeloid cells and B lymphocytes(13, 14) . The function of each of these kinases in cell signaling and differentiation is not precisely known, but an important role is indicated by the fact that btk mutations in both humans and mice result in abnormal B cell development(13, 14, 17, 18) .

In this report we describe a protein tyrosine kinase isolated from embryonic thymus termed Rlk (for Resting lymphocyte kinase), whose structure reveals that it is a novel member of the Tec/Itk/Btk family of protein tyrosine kinases. Like Itk, Rlk is expressed preferentially within the T cell lineage and is present in the thymus as well as in resting mature peripheral T lymphocytes. However, the amino-terminal sequence of Rlk diverges from that of Itk and the other subfamily members, suggesting that it may play a distinct role in T cell activation and thymocyte maturation.


MATERIALS AND METHODS

Cells and Antibodies

The monocytic cell line 162R, the myeloblastic line M1, the B cell lymphoma WEHI 231, the B cell line BCL1 and the transformed cell line originally of cytotoxic T cell origin BFS, the pre-B cell line 70Z/3, and a normal T helper (T(H)) cell line were provided by Drs. Luca Gusella, Scott Durum, Chou-Chi Li, Howard Young, Sergei Nedospasov, and Dennis Taub, respectively, all of the Biological Response Modifiers Program, National Cancer Institute, Frederick, MD. CTLL T cells were a gift of Dr. T. Yi, Cleveland Clinic, Cleveland, OH. The helper T cell clone HT-2.A5E, the thymic lymphoma WEHI 7.1, and the B cell lymphoma RAW 8.1 were purchased from ATCC (Rockville, MD). Ovalbumin-specific Th1 (pGL2, pGL10, J619) and Th2 (pL3, pL104, D10) CD4 T cell clones were provided by Dr. Frank Fitch, University of Chicago, Chicago, IL. Bosc-23 cells (19) were the gift of Drs. W. Pear and D. Baltimore (Rockefeller University, New York, NY).

The rabbit polyclonal antiserum was raised against a synthetic peptide spanning amino acid residues 505 to 527 of Rlk. Peptide at 5 mg/ml was coupled to ovalbumin (Sigma) with 0.2% glutaraldehyde at a ratio of 50:1. Immunizations in complete Freund's adjuvant were carried out at 14-day intervals. Preimmune and immune antisera were purified on a peptide column utilizing Rlk coupled to epoxy-activated cross-linked 4% beaded agarose (Sigma).

DNA and RNA Analyses

The construction of a day 16 murine embryonic thymus library, isolation of total library cDNA by helper phage rescue, polymerase chain reaction (PCR) (^1)amplification using degenerate oligonucleotide primers corresponding to conserved regions in the catalytic domain of protein tyrosine kinases, cloning and sequencing of amplified products, and library screening and sequencing of cDNA clones were all performed as described previously(20) .

For Northern analyses, RNA was isolated from various tissues or cell lines by the guanidine isothiocyanate method(21) . Total cellular RNA (20 µg) was fractionated by electrophoresis in agarose-formaldehyde gels according to standard protocols, transferred to nitrocellulose, and hybridized at 68 °C for 2-3 h in QuickHyb solution (Stratagene) to a P-labeled 500-base pair SmaI-KpnI fragment of the rlk cDNA clone. The same filters were hybridized to P-labeled probes from the chicken beta-actin and human G3PDH genes as controls. Analysis of rlk expression by reverse transcriptase PCR was performed using the preamplification system from Life Technologies, Inc. Oligo(dT)-primed cDNA synthesized from 1 µg of total cellular RNA was amplified using primers specific for rlk (upstream, 5`-GAAACGTGGTGACCTTAAATGC-3`; downstream, 5`-AGGTTGGTGTGGAGCATA CTCT-3`) expected to generate a 300-base pair fragment. Thirty cycles were run according to the following protocol: 95 °C for 1 min, 57 °C for 2 min, and 72 °C for 3 min. beta-actin primers were used to generate control cDNA products.

To express the rlk cDNA, a NheI-ApaI fragment encompassing the entire coding region was subcloned into pGEM11ZF (Promega). A single Myc epitope tag (22, 23) (EQKLISEEDL) was joined to the Rlk protein by ligating a double-stranded synthetic oligonucleotide having the sequence 5`-CATGGAGCAGAAGCTCATCTCAGAAGAAGACCT-3` to an NcoI site upstream of the predicted start ATG of rlk, thereby fusing the Myc tag in frame to the amino terminus.

Protein Analyses

A HindIII-BamHI fragment from the Myc epitope tagged rlk cDNA was subcloned into the retroviral vector pLNCX(24) . Two control plasmids, CA10WT9E10, encoding the first 251 amino acids of c-Src followed by a Myc tag, and LNCX-src, encoding wild type c-Src, were gifts of Drs. K. Kaplan and H. E. Varmus (UCSF). Bosc-23 cells were transfected by CaPO4-Bis with 10 µg of plasmid DNA as described previously(25) . Forty-eight hours after transfection, viral supernatants were collected, the cells were washed twice in ice-cold phosphate-buffered saline, and lysed in Lysis Buffer (0.5% Nonidet P-40, 50 mM Hepes, pH 7.0, 5 mM EDTA, 50 mM NaCl, 10 mM NaPo(4), pH 7.2, 50 mM NaFl with 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml pepstatin, and 100 µg/ml aprotinin) as described previously(17) . Lysates were spun for l5 min in a microcentrifuge at 4 °C and the supernatants were precleared with protein A-Sepharose (Pharmacia BIotech, Inc.) and then incubated with Myc epitope-specific monoclonal antibody 9E10 (Oncogene Science) overnight. Protein A-Sepharose preadsorbed with rabbit anti-mouse serum (Jackson ImmunoResearch Laboratories) was added for an additional 2 h and then samples were washed 2 times each with ice-cold Wash Buffer (phosphate-buffered saline, 0.01% Brij-35) and Kinase Buffer (50 mM Hepes, pH 7.0, 0.1 mM EDTA, 0.1 mg/ml bovine serum albumin, 0.15 M NaCl, 10 mM dithiothreitol, 0.01% Brij-35, 1 mM sodium vanadate). Samples were resuspended in 40 µl of Kinase Buffer and divided in half. To one half of the sample, MgCl(2) (50 mM) and cold ATP (0.1 mM) were added to initiate the kinase reaction. To the other half, 16 µCi of [- P]ATP was also added. After 20 min, the reaction was terminated, and excess ATP was removed by washing twice with ice-cold Wash Buffer. Samples were separated on 10% SDS-polyacrylamide gel electrophoresis. Gels containing radioactive labeled samples were dried for autoradiography. Nonradioactive samples were electrotransferred to nitrocellulose for detection with 4G10 monoclonal anti-phosphotyrosine antibody (Upstate Biotechnology Inc.) according to the manufacturer's protocols. Detection was performed with ECL reagents (Amersham Corp.) Blots were then stripped and reprobed with 9E10 to confirm immunoprecipitation with the anti-Myc antibody. For phosphoaminoacid analysis, in vitro kinase reactions were performed as above, samples were electrophoresed by SDS-polyacrylamide gel electrophoresis, and transferred to Immobilon (Millipore). Bands were excised and subjected to phosphoaminoacid analysis by thin layer chromatography in one dimension as described previously(26) .

Fresh murine tissue samples were lysed for 30 min in Lysis Buffer (0.5% Triton X-100, 5 mM EDTA, 50 mM Tris base, pH 7.6, 150 mM NaCl, 1 mM Na(3)VO(4), 10 µg/ml leupeptin, 10 µg/ml aprotinin, 25 µMp-nitrophenyl p`-guanidinobenzoate, 1 mM phenylmethylsulfonyl fluoride), and insoluble cellular debris was removed by centrifugation. The samples were precleared with 100 µl of JB610 hyperimmune rabbit serum (raised against an irrelevant class I major histocompatibility complex peptide) and precipitated twice with 100 µl of a 50% slurry of protein A-Sepharose. Ten micrograms of column-purified Ig was then incubated with 60 µl of 50% protein A-Sepharose at 4 °C for 1 h. The beads were then washed and mixed with the cell lysates for 12 h at 4 °C. The amounts of protein from different tissues were equalized for the immunoprecipitations using the Bio-Rad protein Assay (Bio-Rad). The immunoprecipitation reactions were washed twice with Lysis Buffer, twice with the same buffer lacking EDTA, and Triton X-100 and then incubated for 15 min at room temperature with 60 µl of Kinase Buffer (20 mM HEPES, 100 mM NaCl, 5 mM MgCl(2), 1 µM ATP, 5 mM MnCl(2), 10 µCi [P]ATP). The reaction was stopped with 20 mM EDTA Lysis Buffer and washed 3 times with 5 mM EDTA Lysis Buffer. Immunoprecipitates were eluted by boiling for 5 min in reducing sample buffer (1 M Tris, pH 6.8, 10% SDS, 10% glycerol, 5% 2-mercaptoethanol) followed by vortexing and then electrophoresed on a 10% polyacrylamide gel. Products were detected by autoradiography for various periods at -80 °C using an intensifying screen. Peptide blocking was done with a 20-fold molar excess of either Rlk 505-527 or the heterologous peptide PRQHHSCRFDGTRNQGFCV without washing the beads prior to the addition of the cell lysates. For the peptide blocking, the experimental conditions were modified to reduce background and enhance specificity as follows. Immunoprecipitation was for 6 rather than 12 h; 10 mM NaF was added to the lysis buffer; 0.3 M NaCl was used in the two washes prior to the kinase reaction; the kinase reaction was performed at 4 °C for 15 min; samples were not precleared.


RESULTS

Cloning and Sequencing of rlk cDNAs

A PCR-based strategy was employed in order to identify protein tyrosine kinases of potential importance in early T cell development. Degenerate primers corresponding to conserved sequences in the kinase domain of protein tyrosine kinases were used to amplify total cDNA derived from a day 16 murine embryonic thymus cDNA library, and the products of anticipated size were cloned and sequenced. One clone represented a novel kinase with greatest homology to the Tec kinase and was used to screen the fetal thymus cDNA library.

Several independent cDNA clones were sequenced that appeared to encompass a complete reading frame of the same protein. One of these clones, K9.D, contained an open reading frame of 1581 nucleotides encoding a protein of 527 amino acids with a predicted molecular mass of 62 kDa (Fig. 1). An in frame methionine was encoded by nucleotides 49-51 in the context of a consensus sequence for translation initiation(27) . Rapid amplification of cDNA ends PCR analysis indicated that this AUG corresponded to the first AUG of the majority of mRNAs analyzed. (^2)The predicted amino acid sequence revealed certain characteristic features of tyrosine kinases(28) , including an SH3 domain (residues 89-136), a single SH2 domain (residues 150-246), and sequence motifs (DLAARN, residues 390-395; PVKWCPPE, residues 429-436) that are distinctive for non-Src subfamily protein tyrosine kinases (Fig. 1). Other residues conserved in protein kinases were identified in the sequence (Fig. 1), including the tyrosine (Tyr-420) that is a site of autophosphorylation in Src-related kinases(28) . Because of its pattern of expression (see below), we have designated this gene rlk (for resting lymphocyte kinase).


Figure 1: Primary structure of rlk. Complete nucleotide sequence of the rlk cDNA and the deduced amino acid sequence. Nucleotide numbers are shown at the left and the corresponding amino acid numbers at the right. The SH3, SH2, and kinase domains are bracketed by arrows. Residues that constitute the ATP binding site are circled. The invariant lysine (K) of the phosphotransferase domain and the autophosphorylation site (Y) are boxed. The conserved DFG as well as the conserved sequences corresponding to the PCR primers are underlined.



The predicted sequence of the Rlk kinase exhibited strongest homology to the proteins of the recently characterized group of tyrosine kinases that include Btk, Itk, and Tec (Fig. 2). Like the other members of this subfamily, Rlk lacks the consensus amino-terminal myristylation signal (Gly at position 2, Lys or Arg at position 7) as well as the negative regulatory carboxyl-terminal tyrosine conserved in Src family kinases(29, 30) . Rlk displayed similar levels of homology to the other kinases in this subfamily among all of the shared domains (Fig. 2B).


Figure 2: Amino acid sequence of Rlk in relation to tyrosine kinases of the same subfamily. A, comparison of the predicted amino acid sequence of Rlk with those of the closely related proteins Tec, Tec2, Itk, and Btk. The conserved residues are boxed. Gaps were introduced to optimize the alignment. B, percentage amino acid identity between Rlk and closely related protein tyrosine kinases. The percent identity is shown for the SH3, SH2, and kinase domains as well as the degree of overall identity.



However, the sequence of Rlk was found to be divergent within the amino-terminal domain. (Fig. 2A). The Btk, Itk, and Tec kinases have extended amino termini (nearly 200 amino acids) upstream of SH3, with a significant degree of sequence identity (>40%) that includes a pleckstrin homology domain(31) . In contrast, Rlk has a unique amino-terminal sequence of 88 amino acids with no significant similarity to any other sequence in the data base.

Tissue-specific Expression of rlk mRNA

Because the other members of this kinase subfamily all manifest highly restricted patterns of expression within distinct tissue and cell types, we determined whether rlk, originally derived from fetal thymus, would be expressed predominantly within the T lymphocyte lineage.

Northern blot analysis was performed on various murine tissues using a probe derived from a KpnI-SmaI fragment of rlk encompassing 500 base pairs of the coding region. A major rlk transcript of 2.3-2.4 kilobase pairs was abundant in both day 16 embryonic and adult thymus and was also present in spleen and lymph node (Fig. 3, A and B). A low level of rlk mRNA was also detected in testis. Rlk message was absent from heart, liver, kidney, and brain. Minimal expression was variably observed in the lung, but the absence of any rlk expression in the lungs of athymic (nu/nu) mice (Fig. 3B) indicated that this was probably due to contamination with hilar lymph nodes. Consistent with predominant expression in the T cell lineage, only very low levels of rlk mRNA were detected in the spleens of young athymic mice, which are populated by B cells but have very low numbers of T lymphocytes (Fig. 3C). Stimulation of nude mouse spleen cells with the B cell mitogen lipopolysaccharide failed to induce rlk message (Fig. 3C). The low but perceptible rlk expression in nude spleen could reflect the presence of natural killer (NK) cells or alternatively low level rlk expression in some B cell subsets or at certain stages of B cell development.


Figure 3: Northern blot analyses showing the pattern of rlk mRNA expression in various murine cells and tissues. The sizes of the hybridizing transcripts in kilobases are shown on the left. Equivalent levels of mRNA in each sample were determined by expression of beta-actin and G3PDH (right) mRNAs. A and B represent separate experiments assessing rlk expression in independently prepared samples from different tissues. C, Rlk and beta-actin mRNA in cell lines and spleen from nude (athymic) mice. 162R, monocytic cell line; M1, myeloblastic cell line; WEHI231, BCL1, B cell lymphoma and cell line; BFS, long term in vitro cultured transformed cell line originally of CD8 cytotoxic T cell origin. Normal T(H) line is an in vitro cultured, phenotypically normal antigen-specific CD4 T cell population.



While abundant in freshly isolated adult thymus and mature peripheral resting T lymphocytes from spleen and lymph nodes, rlk was also found at significant levels within embryonic thymus as early as day 13 of gestation, at which time thymocytes all bear an immature CD4 CD8 surface phenotype (Fig. 3B)(32) . Taken together, these results indicated that rlk expression is present early in thymocyte maturation, is maintained throughout development, and remains restricted predominantly to the T cell lineage. This was further supported by analysis of various B cell, macrophage, and myelocytic cell lines, all of which failed to express any rlk message (Fig. 3C). Moreover, three additional T cell lines, the cytotoxic line CTLL, the helper line HT-2, and the thymic lymphoma WEHI 7.1 were all positive for rlk, while the pre-B cell line 70Z/3 and the B cell lymphoma RAW 8.1 were negative (data not shown). Interestingly, one T lineage-derived line, BFS, did not express detectable rlk mRNA. Originally a cytotoxic T cell line, BFS has undergone prolonged culture and, in addition to acquiring a transformed phenotype, has lost surface phenotypic characteristics of T lymphocytes.

In order to examine further the expression of rlk in peripheral T cell subsets, the presence of rlk transcripts was evaluated in various antigen-specific T cell clones. Interestingly, this analysis indicated that rlk message was reproducibly far more abundant in Th1 as compared with Th2 phenotype CD4 helper T cell clones (Fig. 4). These helper T cell subsets are distinguished by their patterns of lymphokine production upon activation such that Th1 T cells produce interleukin-2 and interferon-, whereas Th2 clones secrete interleukin-4, interleukin-5, transforming growth factor beta, and interleukin-10(33) . Rlk transcripts accumulated at significantly greater levels in Th1 clones (15-20-fold), although very low level rlk expression could be detected by RNA PCR analysis in some Th2 clones (data not shown).


Figure 4: Preferential expression of rlk in Th1 relative to Th2 T cell clones. Reverse transcriptase PCR was performed on RNA from various antigen-specific T cell clones using rlk-specific primers or beta-actin specific primers as described under ``Materials and Methods.'' The amplification products were electrophoresed in 1% agarose and then stained with ethidium bromide. Lanes1-3, Th2 clones D10, PL3, and PL104; lanes4-6, Th1 clones PGL2, PGL10, and J619; lane7, heart RNA, negative for rlk, positive for beta-actin; lane8, day 16 embryonic thymus, positive control. The sizes of the cDNA amplification products are shown at the left.



Tyrosine Kinase Activity and Detection of Rlk Protein

To confirm the enzymatic activity of Rlk, a Myc epitope tag was added to the amino-terminal coding region of rlk, and this construct was expressed in Bosc23 cells. Lysates of rlk-transfected cells were immunoprecipitated with a Myc-specific mAb and were assayed for in vitro kinase activity. A prominent 67-kDa phosphorylated protein was recovered from immunoprecipitation reactions of lysates of cells transfected with the epitope-tagged rlk cDNA (lanes3 and 4), while mock-transfection (lane1) or transfection with a Myc-tagged construct containing only the SH3 and SH2 domains of c-Src (lane2) resulted in no detectable kinase activity (Fig. 5A). The larger size of this protein relative to Rlk is accounted for by the extra amino acids encoded following the fusion of the Myc tag.


Figure 5: Kinase activity and immunoblotting of Rlk expressed in mammalian cells. A, autophosphorylation of Rlk. cDNA constructs in the retroviral vector pLNCX were transfected into the retroviral helper cell line Bosc-23, the lysates were immunoprecipitated with the Myc epitope specific monoclonal antibody 9E10, and in vitro kinase assays were performed in the presence of [-]PATP. Lane1, mock transfected; lane2, transfected with Myc-tagged control construct src-SH3-SH2 lacking a kinase domain; lanes3 and 4, two independent transfections with rlk cDNA in pLNCX. A 67-kDa phosphorylated product is recovered exclusively from the reactions with the rlk transfectants. B, Rlk contains phosphotyrosines. In vitro kinase reactions of the same immunoprecipitates were performed with cold ATP and subjected to phosphotyrosine immunoblotting with the phosphotyrosine-specific antibody 4G10. A 67-kDa tyrosine-phosphorylated protein is again exclusively observed in the rlk transfectants (lanes3 and 4). Ig heavy chain is observed in all lanes. C, phosphorylation of Rlk on tyrosine. Immunoprecipitation and in vitro kinase assay was performed as in A on rlk as well as c-src transfectants, and the products were fractionated by electrophoresis and transferred to an Immobilon filter. The regions containing phosphorylated Rlk or Src and the corresponding region in the control lane were excised and subjected to phosphoamino acid analysis by thin layer chromatography in one dimension. Lane1, c-Src; lane2, Rlk; lane3, control. Positions of phosphoserine (S), phosphothreonine (T), and phosphotyrosine (Y) markers are identified. In the control lane, only the cold phosphotyrosine standard was included.



We next immunoblotted the Rlk protein immunoprecipitated from the cell lysate with anti-P-Tyr antibody following an in vitro kinase reaction. An approximately 67-kDa tyrosine phosphorylated product was demonstrated exclusively with the immunoprecipitates of the rlk-transfected cells, in contrast to mock- or src SH3-SH2-Myc-transfected cells (Fig. 5B). Western blotting with the mAb against the Myc epitope tag confirmed that the 67-kDa protein represented Rlk (data not shown). Finally, phosphoaminoacid analysis of the P-labeled 67-kDa protein confirmed that the phosphorylation of Rlk had occurred on tyrosine (Fig. 5C).

In order to detect Rlk in normal T lymphocytes, and to examine the kinase activity of this protein, a polyclonal antiserum was raised against a peptide corresponding to amino acid residues 505-527 of the Rlk protein. The anti-Rlk antibody was incubated with extracts of murine thymus, lymph node, kidney, and liver, and the immunoprecipitates were assayed for protein kinase activity (Fig. 6A). A 62-kDa phosphorylated protein was recovered from the in vitro kinase reactions performed on the immunoprecipitates of lymph node and thymus (Fig. 6A, lanes3 and 4), but not liver or kidney (Fig. 6A, lanes1 and 2). Immunoprecipitations and in vitro kinase reactions were repeated on thymus extracts with the anti-Rlk antiserum in the presence of a 20-fold molar excess of peptide 505-527 as well as a heterologous peptide (Fig. 6B). A 62-kDa phosphorylated protein was detected in the presence of excess heterologous peptide (lane3) but not in the reaction performed in the presence of the homologous Rlk peptide inhibitor (lane4). No phosphorylated protein was recovered from the reactions with preimmune rabbit IgG in the presence of either peptide (lanes1 and 2).


Figure 6: Immunoprecipitation of Rlk from cells and tissues and phosphorylation in vitro. A, Rlk kinase activity is restricted to lymphoid organs. Lysates from various tissues were immunoprecipitated with a rabbit antiserum raised against a peptide spanning residues 505-527 of Rlk, and in vitro kinase assays were performed as described under ``Materials and Methods.'' Equal protein loading was verified as described under ``Materials and Methods.'' Exposure was for 24 h. Lane1, liver; lane2, kidney; lane3, lymph node; lane4, thymus. A 62-kDa phosphorylated product is found exclusively in lymph node and thymus (arrow at right). B, immunoprecipitation and in vitro kinase assay in the presence of competitor peptides. Immunoprecipitation of thymocyte extract was performed with preimmune antibody (lanes1 and 2) or anti-Rlk peptide antibody (lanes3 and 4) in the presence of 20-fold molar excess of heterologous (lanes1 and 3) or homologous (lanes2 and 4) competitor peptides. The exposure time was 6 days. Molecular mass markers (kDa) are shown at left.




DISCUSSION

We have characterized Rlk, a cytoplasmic tyrosine kinase expressed predominantly in cells of the T lymphocyte lineage, which represents a novel member of a recently identified subfamily of Src-related enzymes that includes Itk, Btk, Tec, and Tec2. Like the Itk, Btk, and Tec2 proteins, Rlk is expressed in a tissue-specific fashion predominantly within a hematopoietic lineage (Tec expression is maximal in hepatocytes). Also in common with the other members of this subfamily, Rlk is capable of autophosphorylation but lacks the N-terminal myristylation signal as well as the negative regulatory C-terminal tyrosine characteristic of Src kinases.

However, Rlk is distinguished from Itk, Btk, and Tec by its divergent amino terminus. The region of homology shared by the former three proteins has been reported to include an apparent pleckstrin homology domain. Because a pleckstrin homology sequence in the beta-adrenergic receptor kinase corresponds to a heterotrimeric G-protein binding region, a possible role for this domain in signal transduction has been suggested(31) . Evidence consistent with a signaling role for the amino-terminal domain is provided by the X-linked immunodeficient (xid) mouse strains, which harbor a btk gene encoding an amino-terminal missense mutation, thus altering an evolutionarily conserved arginine at amino acid position 28(17, 18) . The mutant murine Btk protein, although expressed at normal levels and retaining kinase activity, must be functionally abnormal in view of the phenotypic immaturity of B cells in animals bearing the xid mutation. B cells from xid mice are characterized by an inability to respond to thymus-independent polysaccharide antigens, high surface IgM to IgD ratios, and abnormal proliferation in response to activation signals such as Ig receptor cross-linking(34) . In this light, the disparate amino-terminal sequence of Rlk could determine a unique set of intermolecular interactions with distinct substrate specificity or intracellular localization.

The expression of Rlk early in thymic ontogeny suggests that it may function in modulating T cell development. Several genetic studies have confirmed a role for protein tyrosine kinases during intrathymic T cell differentiation. Thus, a subset of thymocytes from fynT knockout mice have T cell receptor-mediated signaling abnormalities (35, 36) . Lck-deficient mice have severe thymic atrophy and a block in thymocyte maturation to the CD4CD8 phenotype (37) . Individuals with selective T cell defect immunodeficiency express a kinase negative variant of ZAP-70, which results in an absolute deficiency of CD8 peripheral T cells and severely impaired mitogenic signaling in the CD4 subset(38) .

The phenotypes of xid mice and XLA patients demonstrate that the Btk/Itk/Tec subfamily of kinases also have a role in hematopoietic development. XLA patients exhibit mutations that either abolish transcription of the btk gene or alter the sequence of the kinase domain, resulting in a complete loss of enzymatic function(13, 14) . Consequently they display a more abnormal phenotype than xid mice, with a complete block in B cell development at an immature pre-B cell stage. This results in a lack of circulating mature B cells and serum immunoglobulins of all isotypes, and enhanced susceptibility to recurrent bacterial as well as viral and parasitic infections.

Given the restricted expression of Itk in the T cell lineage and its homology to Btk throughout all regions of the protein, it seems possible that Itk plays a role in T cell development analogous to that of Btk in B cells. In contrast, the divergent unique amino-terminal domain of Rlk suggests a role in T cell signal transduction distinct from Itk. Moreover, in comparison to Itk(11, 12, 39) , Rlk appears to be expressed at more significant levels in mature resting peripheral T cells relative to the high levels in the thymus ( Fig. 3and Fig. 6), although this remains to be quantitated precisely. Therefore, Rlk and Itk may well subserve complementary, although independent roles in T cell development and activation, perhaps analogous to the distinct functions in T cell signal transduction that are mediated by the related Src kinases Lck and FynT(35, 36, 37, 40) .

Although Rlk could function in signal transduction through the T cell receptor, different patterns of protein tyrosine phosphorylation also follow the engagement of other T cell costimulatory surface molecules such as CD2 and CD28, and the kinases that regulate these signaling pathways remain to be determined(41, 42, 43) . It is possible that Rlk could play a role in T cell activation through these costimulatory cell surface molecules.

Finally, the observation that Rlk is preferentially expressed in Th1 phenotype relative to Th2 phenotype T cells suggests that Rlk could mediate intracellular signaling following T cell stimulation by cytokines. Alternatively, Rlk could function in signal transduction pathways leading to the selective transcriptional up-regulation of particular cytokine genes.

Following the submission of this manuscript, Haire et al.(44) reported the cloning of a gene derived from peripheral blood mononuclear cells, TXK, that represents the human homologue of rlk. Like rlk, TXK is expressed predominantly in T cells, although a myeloid cell line was also found to express the gene. The predicted amino acid sequences of the murine and human genes are highly conserved, with an overall identity of 83%.


FOOTNOTES

*
This work was supported in part by Alexion, Inc. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' 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 GenBank(TM)/EMBL Data Bank with accession number(s) L35268[GenBank].

§
Contributed equally to this work.

Supported by National Institutes of Health Endocrinology Training Grant DK07011.

**
Supported by a Leukemia Society special fellowship.

§§
To whom correspondence and reprint requests should be addressed: Alexion Pharmaceuticals, 25 Science Park, Ste. 360, New Haven, CT 06511. Tel.: 203-776-1790; Fax: 203-772-3655.

(^1)
The abbreviations used are: PCR, polymerase chain reaction; xid, x-linked immunodeficiency; XLA, x-linked agammaglobulinemia.

(^2)
P. Schwartzberg and M. Chamorro, unpublished observations.


ACKNOWLEDGEMENTS

We thank R. Wange and G. H. Donovan for assistance with phosphoamino acid analysis.


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