(Received for publication, June 15, 1994; and in revised form, November 8, 1994)
From the
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.
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.
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).
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
-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.
-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.
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 NaVO
, 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
, 1 µM ATP, 5 mM MnCl
, 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.
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. ()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.
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 -actin and G3PDH (right) mRNAs. A and B represent separate experiments assessing rlk expression in independently prepared samples from different
tissues. C, Rlk and
-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
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
, 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 -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
-actin; lane8, day
16 embryonic thymus, positive control. The sizes of the cDNA
amplification products are shown at the left.
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.
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 -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%.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L35268[GenBank].