(Received for publication, July 26, 1995; and in revised form, September 1, 1995)
From the
The human leukemic Jurkat cell line is commonly used as a model cellular system to study T lymphocyte signal transduction. Various clonal derivatives of Jurkat T cells exist which display different characteristics with regard to responses to external stimuli. Among these, the E6-1 clone of Jurkat T cells has been used as a parental line from which numerous important somatic mutant clones have been generated. During the course of experiments examining signals initiated by the T cell antigen receptor in an E6-1-derived Jurkat cell clone J.CaM1, we observed that the 72-kilodalton Syk protein tyrosine kinase previously found in other Jurkat cells was not detected. Upon further analysis it was determined that Syk transcripts from the J.CaM1 cells as well as the parental E6-1 cells contain a single guanine nucleotide insertion at position 92. This nucleotide insertion results in a shift in the Syk open reading frame leading to alternate codon usage as well as the generation of a termination codon at position 109. Thus, Syk transcripts in E6-1 cells and E6-1-derived clones are predicted to be capable of encoding only the first 33 amino acids of the 630-amino acid wild type Syk. These findings are incompatible with a recently proposed model of T cell antigen receptor signal transduction based, in part, on experiments conducted using E6-1-derived cells, suggesting that Syk might play a role upstream of Lck and Zap70.
The Syk protein tyrosine kinase together with the Zap70 protein
tyrosine kinase comprise a family of cytoplasmic enzymes that are
important for signal transduction initiated by different types of
surface receptors in cells of hemopoietic origin(1) . Unlike
other cytoplasmic protein tyrosine kinases, Syk and Zap70 possess
tandem SH2 domains amino-terminal to their catalytic
domain(2, 3) . The Syk and Zap70 SH2 domains serve to
bind tandem phosphotyrosine containing elements in the
membrane-associated signal coupling subunits of immune recognition
receptors(1) . These 18-20 amino acid elements are
referred to as immunoreceptor tyrosine activation motifs and have the
consensus sequence
(D/E)XXYXX(I/L)XYXX(I/L) (4) . Association of Syk and Zap70 with the phosphorylated
immunoreceptor tyrosine activation motifs serves to position the
kinases in a membrane proximal location and contributes to the
activation of the
enzymes(5, 6, 7, 8, 9, 10) .
Zap70 is expressed in all major thymocyte populations as well as in
mature T cells of both CD4 and CD8
lineages(11) . Syk too is expressed in all of the major
thymocyte populations although the levels of Syk diminish severalfold
in peripheral T lymphocytes(11) . Syk is also expressed in an
additional number of hemopoietic cells including B
cells(11, 12) , mast cells(13, 14) ,
neutrophils(15) , macrophages(16) , erythroid
cells(17) , and platelets(18) . Current evidence
suggests that Zap70 and Syk may potentially contribute in distinct ways
to signal transduction events in T cells and thymocytes. Patients with
mutations in the ZAP gene demonstrate abnormal development of
thymocytes leading to the production of exclusively CD4
T cells in the periphery(19, 20, 21) .
These T cells are, however, unresponsive to mitogen and antigen
stimulation(19, 20, 21) . While no SYK
mutations have been documented in humans, targeted disruption of the syk gene in mice has been shown to block B cell but not T cell
developmental pathways(22) .
Much of our understanding of
the signaling events initiated following the surface engagement of the
T cell antigen receptor (TcR) ()has been based upon model
cell systems. One of the most useful and widely studied of these T cell
models has been the human T cell leukemia line Jurkat. Indeed, Zap70
was initially identified as a TcR
subunit-associated protein and
molecularly cloned from Jurkat T cells(2, 23) .
Previous studies have demonstrated that Jurkat T cells express both Syk
and Zap70(8, 11) . Following TcR cross-linking both
Syk and Zap70 were found to be enzymatically activated in a temporally
indistinguishable manner (8) and both were found to be capable
of association with the TcR
subunit(8, 11) .
During the course of experiments examining protein tyrosine kinase
signaling in the J.CaM1 somatic cell mutant isolated from the Jurkat
E6-1 line(24) , we noticed that the 72-kDa Syk protein tyrosine
kinase was not readily detected. On further analysis it was found that
J.CaM1, the parental Jurkat E6-1 line, as well as another E6-1-derived
cell line J45.01(25) , express Syk transcripts containing a
single guanine nucleotide insertion at position 92 which results in a
frameshift leading to premature termination of the Syk open reading
frame at position 109. In contrast, all Syk transcripts from other
Jurkat lines in which p72
was readily detected
were found to encode wild type Syk. These results demonstrate that
functional Syk is not expressed in all Jurkat T cell-derived clones.
Figure 1:
Expression of
p70 and p72
in Jurkat
cell clones. Detergent lysates of the indicated Jurkat clones were
adjusted to 1 mg/reaction and immunoprecipitation and immunoblot
analysis of either Zap70 (left panel) or Syk (right
panel) performed. The lane marked C in each panel
represents H33HJ lysates immunoprecipitated with preimmune rabbit sera.
The positions of prestained molecular mass markers in kilodaltons (Life
Technologies, Inc.) are indicated.
Figure 2:
Northern blot analysis of Syk transcripts
in Jurkat cell clones. Ten µg of poly(A) RNA
isolated from the indicated Jurkat cell clones was analyzed using a
full-length Syk cDNA (upper panel) or
-actin (lower
panel) probe. The positions of RNA molecular size markers (Life
Technologies, Inc.) in kilobases are shown on the right while
the positions of 28 S and 18 S ribosomal RNAs are shown on the left of each panel.
Figure 3: Syk nucleotide sequence and deduced amino acid sequences from Jurkat cell clones. The nucleotide sequence derived from representative cDNAs from H33HJ (J) and E6-1 (E6) Jurkat cell clones are indicated. The corresponding amino acids predicted from the cDNA sequences are given below the nucleotide sequences with the H33HJ amino acid sequences above those predicted for the E6-1 amino acid sequences. The numbers on the left refer to nucleotide positions within the open reading frame. The Syk sequence derived from the H33HJ cells is the same as that previously determined from cDNAs isolated from Daudi Burkitt's lymphoma cells(30) .
The consequence of the guanine insertion at position 92 predicts that the Syk open reading frame should be shifted and undergo premature termination at position 109 allowing for the potential production of an Syk peptide of only 35 amino acids. This severely truncated gene product would not be detectable with our Syk antisera. In keeping with this prediction, the results shown in Fig. 4demonstrate that transcription/translation of the Syk open reading frame from the E6-1-derived cells failed to produce a detectable Syk protein while the Syk open reading frame obtained from non-E6-1-derived Jurkats produced the expected 72-kDa Syk protein.
Figure 4:
Transcription/translation of cDNAs derived
from Jurkat cell clones. One µg of plasmid DNA from the indicated
Jurkat cell clones was used for transcription/translation in
TNT-coupled rabbit reticulocyte lysates. The translation products were
diluted in cell lysis buffer, immunoprecipitated with anti-Syk, and
immune-complex protein kinase assays conducted. The positions of
p72 (Syk) and prestained molecular mass
markers are indicated.
The results presented in this report demonstrate that Jurkat cell clones derived from the Jurkat E6-1 clone fail to detectably express a functional Syk protein tyrosine kinase. The absence of detectable Syk in these cells is at least in part the consequence of a guanine nucleotide insertion at position 92 in the Syk open reading frame. The resulting frameshift allows for alternative usage of two codons before directing the premature termination of the open reading frame at position 109. The mutated Syk open reading frame allows for the predicted translation of the first 33 amino acids of Syk followed by histidine and glutamic acid prior to termination. While it is as yet unclear whether this predicted Syk amino-terminal peptide is expressed in the E6-1-derived cells, it is not anticipated that this peptide would be capable of any function ascribed to Syk since it would contain only the first 19 amino acids of the amino-terminal SH2 domain.
We have not analyzed the SYK genomic sequences corresponding to the mutation site observed in the Syk cDNAs isolated from the E6-1-derived cells. Therefore, it is not as yet determined if the nucleotide insertion is located in one or both SYK alleles. While all of the E6-1-derived Syk cDNAs isolated contained the mutation, we have detected very low levels of Syk in some E6-1-derived cells on rare occasions. Thus, our data is most compatible with the idea that a single SYK allele containing this mutation is predominantly expressed in these cells and that the other allele is transcriptionally impaired.
The lack of Syk expression in the E6-1 Jurkat cells does not appear
to significantly alter the TcR-mediated responses of these cells when
compared with Jurkat clones expressing Syk and Zap70. However, we
cannot rule out that the absence of Syk in other E6-1-derived Jurkat
clones might influence their signaling properties. The absence of Syk
expression in E6-1 cells in fact may have facilitated the initial
identification and characterization of Zap70 as the major
-associated protein (2, 23) since Syk would have
also been found associated with
in Jurkat clones capable of
expressing both Syk and Zap70(8, 11) . Moreover, these
results, together with those obtained with mice deficient in Syk
expression (22) as well as other studies analyzing E6-1 Jurkat
signaling(2, 3, 7, 11, 23, 24, 25) ,
argue against a previously proposed model placing Syk upstream of Lck
and Zap70(31, 32) .