Download During Cytokinesis Light Chain, Tctex

Interaction of p59fyn Kinase with the Dynein
Light Chain, Tctex-1, and Colocalization
During Cytokinesis
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of June 18, 2017.
Kerry S. Campbell, Suzanne Cooper, Mark Dessing, Sol
Yates and Annie Buder
J Immunol 1998; 161:1728-1737; ;
http://www.jimmunol.org/content/161/4/1728
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Copyright © 1998 by The American Association of
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References
Interaction of p59fyn Kinase with the Dynein Light Chain,
Tctex-1, and Colocalization During Cytokinesis1
Kerry S. Campbell,2 Suzanne Cooper, Mark Dessing, Sol Yates,3 and Annie Buder
P
rotein tyrosine kinase p59fyn (Fyn)4 is a 59-kDa member of
the Src family of nonreceptor protein tyrosine kinases and
is expressed predominantly in hemopoietic and neural tissues. Two distinct isoforms that are products of alternative splicing
of separate seventh exons were originally defined by their selective
expression in hemopoietic (FynT) and brain (FynB) cells (1). Fyn
has been found to associate weakly with invariant chains of both
TCR and B cell Ag receptor complexes in lymphocytes (2–5), and
its enzymatic activity is increased upon cross-linking of either Ag
receptor (6, 7). Fyn overexpression in T cell lines or thymocytes
results in enhanced TCR signaling (8 –10), while FynT-deficient
mice exhibit defects in TCR-stimulated signaling and proliferation
of thymocytes, although more mature T cells exhibit improved
responsiveness (11, 12). Although deficiency of the other major
Src family member expressed in T lymphocytes, p56lck (Lck),
more profoundly disrupts T cell development (13), mice that lack
both FynT and Lck are entirely deficient in mature peripheral ab
T lymphocytes (14, 15). Fyn kinase can efficiently couple both Ag
receptors to intracellular calcium mobilization (11, 12, 16, 17),
which may be mediated partially through direct tyrosine phosphorylation of the inositol 1,4,5-trisphosphate receptor (18). Fyn is also
Basel Institute for Immunology, Basel, Switzerland
Received for publication November 14, 1997. Accepted for publication April
15, 1998.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
The Basel Institute for Immunology was founded and is supported by F. Hoffmann-La Roche Ltd., Basel, Switzerland.
2
Address correspondence and reprint requests to Dr. Kerry S. Campbell, Fox Chase
Cancer Center, 7701 Burholme Avenue, Philadelphia, PA 19111. E-mail address:
[email protected]
3
Current address: Department of Human Retrovirology, Academic Medical Center,
University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands.
4
Abbreviations used in this paper: Fyn, protein tyrosine kinase p59fyn; GST, glutathione S-transferase; Lck, protein tyrosine kinase p56lck; SH, Src homology; LynA,
p56lyn; LynB, p53lyn; ITAMs, immunoreceptor tyrosine-based activation motifs; Blk,
p55blk; pAb, polyclonal Ab; DB, DNA-binding (protein domain); TA, transcriptional
activation (domain).
Copyright © 1998 by The American Association of Immunologists
involved in the function of IL-7 receptors (19, 20) and IL-5 receptors (21) in lymphocytes. Interestingly, Yasunaga et al. (22)
have observed that the kinase also plays a critical role in mitotic
division (cytokinesis) of proB cells. In addition, Ley et al. (23)
have demonstrated significant cytoplasmic localization of Fyn in
lymphocytes. These results strongly suggest that Fyn plays diverse
cellular roles in addition to receptor-proximal signal transduction,
and defining these alternative roles is an important challenge.
Functional diversity between members of the Src family of kinases is mediated by four classical protein interaction domains.
These Src homology (SH) domains are the kinase domain (SH1),
which binds and phosphorylates substrate; SH2, which binds specific phosphotyrosine-containing sequences; SH3, which selectively binds proline rich sequences; and an amino (N)-terminal
domain of 60 to 90 amino acids. Although the majority of Nterminal amino acids are unique for each kinase, the first 10-residue segment contains common elements and has been termed SH4
(24). This SH4 domain facilitates membrane localization through
myristoylation of a glycine and palmitoylation of cysteines on
most Src family members (24 –29). In addition, the SH4 domains
of Fyn and p56lyn (LynA) have been reported to mediate weak
interactions with unphosphorylated immunoreceptor tyrosinebased activation motifs (ITAMs) of lymphocyte Ag receptors (3,
30 –32) and the signaling effectors phospholipase C-g, GAP
(GTPase-activating protein), and MAP (microtubule-associated
protein) kinase (33), indicating that this domain also facilitates
important protein interactions.
Tctex-1 is the product of a gene within the t complex on chromosome 17 of mice, which was originally cloned from sperm
cDNA as a potentially important distorter component of certain
mutant t haplotypes (34). t haplotypes contain multiple conserved
mutated genetic elements within the t complex that are responsible
for various phenotypes of tail length, embryonic lethality, male
sterility, and sperm cell transmission ratio distortion (35). Analysis
of the Tctex-1 protein has been limited (36), until very recently,
when King et al. (37) demonstrated that it is a light chain component of the cytoplasmic dynein complex. Cytoplasmic dynein is a
large microtubule-based multicomponent ATP-dependent motor
0022-1767/98/$02.00
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The protein tyrosine kinase p59fyn (Fyn) plays important roles in both lymphocyte Ag receptor signaling and cytokinesis of proB
cells. We utilized yeast two-hybrid cloning to identify the product of the tctex-1 gene as a protein that specifically interacts with
Fyn, but not with other Src family kinases. Tctex-1 was recently identified as a component of the dynein cytoskeletal motor
complex. The capacity of a Tctex-1-glutathione S-transferase fusion protein to effectively bind Fyn from cell lysates confirmed the
authenticity of this interaction. Tctex-1 binding required the first 19 amino acids of Fyn and integrity of two lysine residues within
this sequence that were previously shown to be important for Fyn interactions with the immunoreceptor tyrosine-based activation
motifs (ITAMs) of lymphocyte Ag receptors. Expression of tctex-1 mRNA and protein was observed in all lymphoma lines
analyzed, and immunofluorescence confocal microscopy localized the protein to the perinuclear region. Analysis of a T cell
hybridoma revealed prominent colocalization of Tctex-1 and Fyn at the cleavage furrow and mitotic spindles in cells undergoing
cytokinesis. Our results provide a unique insight into a mechanism by which Tctex-1 might mediate specific recruitment of Fyn
to the dynein complex in lymphocytes, which may be a critical event in mediating the previously defined role of Fyn in
cytokinesis. The Journal of Immunology, 1998, 161: 1728 –1737.
The Journal of Immunology
Materials and Methods
Mammalian cell lines
Cell lines and their sources were the murine proB cell line 38B9 (Dr. A.
Rolink, Basel, Switzerland); cytotoxic T cell clone CTLL-2 (Dr. J. Garcı́aSanz, Madrid, Spain); COS-7 cells (Dr. B. Imhof, Geneva, Switzerland);
the murine B cell lymphomas K46, A20, and WEHI-231.7; and NIH 3T3
cells transfected with the ZIP-Fyn plasmid to express high levels of p59fyn
protein (45) (all from Dr. J. Cambier, Denver, CO). The fyn-transfected
murine T cell hybridoma N17 (10) was generously provided by Dr. T.
Yamamoto and colleagues (University of Tokyo, Japan). Cells were cultured as previously described (46). Geneticin (G-418; Life Technologies,
Gaithersburg, MD) was supplemented in cultures of COS-7 transfectants
(500 mg/ml) and N17 cells (770 mg/ml).
Antibodies
Anti-Tctex-1 polyclonal Abs (pAb) were generated in rabbits against purified full length murine Tctex-1, which was produced in Escherichia coli
as a GST fusion protein (see below) and cleaved from the GST with human
thrombin (Sigma). The Tctex-1-specific Ab was affinity purified using the
cleaved protein on CNBr-activated Sepharose (Pharmacia, Uppsala, Sweden), eluted with 3.5 M MgCl2 (pH 7.2), dialyzed against PBS, and stored
at 270°C. Anti-Fyn Abs (both from Santa Cruz Biotechnology, Santa
Cruz, CA) were a mAb (sc-434, mouse IgG1; agarose conjugate used for
immunoprecipitation or unconjugated for intracellular staining) and a rabbit pAb (sc-016, unconjugated, used for immunoblotting and intracellular
staining). Anti-FLAG mAb (M2; mouse IgG1; Kodak, Rochester, NY) was
conjugated to CNBr-activated Sepharose for immunoprecipitation. Antib-tubulin mAb was from Boehringer Mannheim (Mannheim, Germany;
mouse IgG2b). Anti-dynein 74-kDa intermediate chain Ab, clone 74.1 (47)
(mouse IgG2b), was purchased from Chemicon (Temecula, CA). The antihuman MHC class I mAb, W6/32, was used as a hybridoma culture supernatant and was generously provided by Dr. Marina Cella (Basel Institute
for Immunology). Rabbit anti-mouse IgG pAb, goat anti-rabbit IgG conjugated with Texas Red or FITC, and goat anti-mouse IgG1 conjugated
with Texas Red were from Southern Biotechnology Associates (Birmingham, AL). Oregon Green 488-conjugated goat anti-rabbit IgG Ab was from
Molecular Probes (Eugene, OR).
Yeast two-hybrid method
A Gal4-based yeast two-hybrid system was generously made available by
Drs. P. Chevray and D. Nathans (Johns Hopkins University, Baltimore,
MD) (48). The pPC62 plasmid encodes the DNA-binding domain of Gal4
(amino acids 1–147) with 39 SalI and NotI restriction sites, which were
utilized to generate “bait” fusion protein constructs. Bait constructs were
generated by PCR from a K46 murine B cell lymphoma cDNA library
(described below); these were the N-terminal domains of Fyn (amino acids
1– 85, a mutated version with lysines 7 and 9 mutated to alanines, and
truncated versions with indicated amino acids), p55blk (Blk; amino acids
1–56), LynA (amino acids 1– 68), p53lyn (lynB; amino acids 1– 47), the
cytoplasmic domain of Ig-b (amino acids 186 –228), and full length
Tctex-1 (amino acids 1–113). The pPC86 plasmid encodes the Gal4 transcriptional activation domain (amino acids 767– 881) and 39 restriction
sites for generation of “prey” fusion protein constructs. A cDNA library
was prepared from 5 mg of poly(A)1-enriched RNA that had been isolated
from the K46 B cell lymphoma using the Poly(A) Quik Kit (Stratagene, La
Jolla, CA). One half of the K46 cells were resting, while the other half had
been stimulated with sheep anti-mouse IgG (Silenus, Hawthorne, Australia; 1 mg/ml) and harvested at time points between 15 min and 6 h. Directionally cloned cDNA was generated with the ZAP-cDNA synthesis kit
(Stratagene) and inserted into EcoRI/SalI-modified pPC86 plasmid (provided by Dr. T. Watanabe, Kyushu University, Fukuoka, Japan). The library contained about 1.2 million original clones with .95% inserts ranging from 200 to 2000 base pair. Additional prey fusion protein constructs
that were generated by PCR into pPC86 were Tctex-1 truncations (amino
acid sequences described in the text), the cytoplasmic domain of Ig-a (amino acids 160 –220), and the N-terminal domains of Fyn (amino acids
1– 85), LynA (amino acids 1– 68), and Lck (amino acids 1– 67). The yeast
reporter strain was HF7c (generously provided by Dr. D. Beach, Cold
Spring Harbor Laboratory, Cold Spring Harbor, NY) with integrated
growth selection (HIS3) and b-galactosidase (lacZ) reporter genes (49).
For cDNA library screening, yeast were first transfected with the bait plasmid (in pPC62) and subsequently transfected with a library (in pPC86)
using lithium acetate and polyethylene glycol (50). Colonies that grew in
the absence of histidine were secondarily screened for b-galactosidase activity (51). cDNA plasmids were released from positive yeast colonies and
cloned as described (52) and sequenced by PCR using the SequiTherm
cycle sequencing kit (Epicentre Technologies, Madison, WI). Transfectants
were streaked on agarose medium lacking histidine to test for growth reporter activation or on medium containing histidine to test for activation of
the lacZ reporter.
Northern blotting
Total RNA (10 mg/sample) was isolated with guanidinium thiocyanate
from cell lines or mouse tissues (C57BL/6 mice from Iffa Credo,
L’Arbresle, France), separated on a 1% agarose gel, and transferred to a
nylon membrane (Hybond-N; Amersham, Arlington Heights, IL). A full
length tctex-1 cDNA sequence probe and a full length b-actin sequence
probe were generated by PCR and labeled with [32P]dCTP using the
Prime-It random primer kit (Stratagene). The membrane blot was hybridized at 65°C and washed at 65°C.
Immunoblotting
Immunoprecipitates or whole cell lysates (lysed directly in Laemmli buffer)
were separated on SDS-PAGE and electrophoretically transferred to
Immobilon-P membranes (Millipore, Bedford, MA). Immunoblotting and
stripping were performed as previously described (53). Abs were rabbit
anti-Tctex-1 (10 mg/ml) or rabbit anti-Fyn pAb (1 mg/ml). Secondary reagent was 125I-labeled protein A (0.5 mCi/ml; Amersham), and proteins
were visualized by autoradiography. It should be noted that detection of
Tctex-1 can be obscured by a background band at 14 kDa in lymphocyte
whole cell lysates when probed with horseradish peroxidase-conjugated
secondary reagents; this result can be avoided by using 125I-labeled
protein A.
Metabolic labeling, protein precipitations, and in vitro kinase
reactions
COS-7 cells were metabolically pulse-labeled in some studies for 15 to 45
min with 5 mCi of [35S]cysteine/methionine (Amersham) in 20 ml of cysteine/methionine-free Dulbecco’s modified Eagle’s medium and chased for
2 to 9 h in normal medium. Cells were lysed for 30 min on ice in 1%
digitonin buffer, 1% Triton X-100 (Surfact-Amps; Pierce, Rockford, IL)
buffer, or RIPA buffer (1% Triton X-100, 0.1% sodium deoxycholate
(Merck, Darmstadt, Germany)), and 0.1% SDS (Bio-Rad, Hercules, CA),
each containing 75 mM NaCl, 10 mM Tris (pH 7.4), 10 mM NaF, 0.4 mM
EDTA, 1 mM Pefabloc SC (Boehringer Mannheim), 2 mM sodium orthovanadate, and 1 mg/ml each of aprotinin, soybean trypsin inhibitor, and
leupeptin (reagents were from Sigma unless otherwise noted). Lysates were
microfuged at 14,000 rpm for 15 min and subjected to precipitation for 2
to 4 h with GST fusion proteins (10 mg/sample) or Abs (2–5 mg/sample)
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unit that plays important roles in intracellular retrograde organelle
transport, membrane trafficking, mitotic spindle localization, and
centrosome separation during mitosis (38 – 40). Tctex-1 has also
recently been shown to be a component in inner arm I1 of flagellar
dynein (41). A human homologue of the tctex-1 gene was reported
to encode 94% protein homology to the murine protein (42), and
a related human gene, “candidate RP3,” encodes a protein with
55% amino acid identity to human Tctex-1 (43). A homologue of
another mouse t complex-encoded protein, Tctex-2, which has limited sequence similarity to Tctex-1, was also recently identified as
a light chain component of flagellar dynein in Chlamydomonas
(44), and a Tctex-1 homologue in the same species exhibits 60%
identity to the mouse protein (41).
To better define functionally important protein interactions with
the unique N-terminal domain of Fyn, we have utilized the yeast
two-hybrid technique. Using this system, we could not demonstrate direct Fyn interactions with the ITAM-containing B cell Ag
receptor components, Ig-a or Ig-b. Upon screening a B cell cDNA
library, however, Tctex-1 was recognized as a strong Fyn-binding
protein in the yeast system, and this interaction was localized to the
first 19 amino acids of Fyn. The validity of this protein-protein
interaction was confirmed by the capacity of a Tctex-1 fusion protein to bind Fyn from cell lysates. Fyn and Tctex-1 were found to
colocalize during cytokinesis in a T cell hybridoma, thereby suggesting that the interaction with Tctex-1 can selectively recruit Fyn
to the dynein motor complex during mitosis of lymphocytes.
1729
1730
Fyn INTERACTS WITH THE DYNEIN LIGHT CHAIN, Tctex-1
FIGURE 1. Tctex-1 can interact specifically
with the N-terminal domain of Fyn. Yeast were
transfected with various combinations of plasmid
constructs encoding the indicated Gal4 DB fusion
proteins and Gal4 TA fusion proteins. Transfectants were subsequently streaked on agarose media containing (1) or lacking (2) histidine. Yeast
transfectants that grow without histidine demonstrate an interaction between the fusion protein
partners that recruits the TA to the reporter gene
and drives its transcription to permit growth. Fusion protein partners were Tctex-1, the N-terminal domains (-NH) of Fyn, Blk, LynA, and LynB,
and the cytoplasmic domains (cyto.) of Ig-a and
Ig-b.
GST and epitope-tagged fusion proteins and protein expression
GST fusion protein constructs of the Fyn N-terminal domain (amino acids
1– 85) and Tctex-1 (amino acids 1–113) were generated by excision of
these cDNAs from pPC62 and pPC86 and ligation into pGEX-4T-2 plasmid (Pharmacia). Other GST fusion constructs were human platelet-derived growth factor receptor kinase insert region (amino acids 698 –797 in
pGEX-3X; provided by Dr. A. Kazlauskas, Harvard University) and cytoplasmic domains of murine Ig-a and Ig-b (3) (provided by Dr. J. Cambier).
E. coli (Top10F9) were transformed with these plasmids and induced for 2
to 4 h with 0.3 mM isopropyl b-D-thiogalactopyranoside as described (3).
Cells were pelleted, and probe sonicated on ice in 10 ml of Tris-buffered
saline (TBS; 10 mM Tris, pH 7.4, 150 mM NaCl) containing Complete
protease inhibitor (Boehringer Mannheim; 1 tablet/50 ml TBS), admixed to
1% Triton X-100 and pelleted at 12,000 rpm for 15 min. Cleared lysate was
precipitated for 2 h to overnight with glutathione-Sepharose 4B beads
(Pharmacia), and beads were washed with 1% Triton X-100 in TBS and
stored at 4°C as a 50% slurry in the same buffer. A C-terminal FLAG
epitope-tagged Tctex-1 construct was created by PCR encoding DYKD
DDDK-stop after the 113-amino acid murine Tctex-1 sequence and cloned
into the mammalian expression plasmid, pcDNA3 (InVitrogen, San Diego,
CA). COS-7 cells (70% confluent) were transfected in 100-mm petri dishes
with 5 mg of plasmid DNA using Dosper liposomal transfection reagent
according to the manufacturer’s protocol (Boehringer Mannheim), and protein expression was assayed two days later. Stably integrated clones were
isolated by limiting dilution. An expression plasmid encoding N-terminal
FLAG-tagged Tctex-1 was also generated (M-DYKDDDDK-), but did not
produce significant levels of protein expression in COS-7.
Immunofluorescence confocal microscopy
Some cells were grown on chamber slides (see Fig. 7A) or allowed to
adhere on poly-L-lysine coated slides (Fig. 7B) before staining. Other
cells were stained in suspension after sorting and subsequently cytospun
onto slides (Fig. 7C). The following staining protocol was utilized,
since it resulted in optimal retention of b-tubulin structure in cells.
Cells were washed in 37°C serum-free Iscove’s modified Dulbecco’s
medium before fixing at 37°C for 10 min with 3% paraformaldehyde (in
PBS, pH 7.0, and 5.4% glucose). Subsequent steps were performed at
room temperature. After washing twice in PBS (pH 7.4), fixed cells
were permeabilized for 30 min in PBS/saponin (PBS with 0.1% saponin
and 1 mM HEPES) and washed again in the same. Cells were blocked
for 30 min in PBS/saponin/BSA (PBS/saponin with 2% BSA) and
washed once in PBS/saponin. Cells were then incubated for 30 to 45
min with optimal concentrations of primary Ab in PBS/saponin/BSA
and washed three times with PBS/saponin. An optimal concentration of
secondary Ab in PBS/saponin/BSA was added for 30 to 45 min, and
cells were washed three times with PBS/saponin, followed by three
washes with PBS. Some cells were fixed, stained with Hoechst 33342
(Molecular Probes), and sorted on a FACS Vantage into G0/G1, S, and
G2/M stages (55) before intracellular Ab staining (Fig. 7C). Coverslips
were mounted with Mowiol (Hoechst, Frankfurt, Germany) or Fluoromount-G (Southern Biotechnology) mounting solution and analyzed
through a 633/1.4 Zeiss Plan-Apochromat lens on a Zeiss Axiovert 100
inverted microscope fitted with a Bio-Rad MRC 1024 laser scanning
confocal imaging system. Images were acquired in accumulation mode,
analyzed using LaserSharp software (Bio-Rad, version 2.1A), and processed with Adobe Photoshop (version 3.0, Adobe Systems, San Jose,
CA). Compensation was rigidly controlled using single-stained cell
samples and/or independent sequential excitation of each fluorescent
dye to assure lack of bleedover between the green channel and the red
channel. Pretreatment of the anti-Tctex-1 pAb preparation with recombinant Tctex-1 protein completely eliminated immunofluorescent reactivity, thereby indicating specificity of the staining.
Results
Identification of Tctex-1 as a Fyn-binding protein
Previous experiments with GST fusion proteins have suggested
that the N-terminal domains of Fyn and LynA can interact directly
with the cytoplasmic domains of the B cell Ag receptor chains,
Ig-a and Ig-b (31). As demonstrated in Figure 1, we could not
detect any direct protein interactions between these kinase N-terminal domains (or LynB; data not shown) and either Ig-a or Ig-b
cytoplasmic domains in a Gal4-based yeast two-hybrid system in
either orientation (DB or TA fusions), nor in a more sensitive
LexA-based system (data not shown; sensitivity described in Ref.
46). All fusion proteins of appropriate size were produced in the
yeast transfectants, however, as assessed by anti-GAL4 immunoblotting (data not shown). Although these results suggest that Fyn
cannot directly interact with Ig-a or Ig-b, one must consider that
many factors can contribute to a negative result in this assay, and
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prebound to glutathione-Sepharose, GammaBindPlus Sepharose, or protein
A-Sepharose 4 Fast Flow beads (Pharmacia). Protein precipitations were
washed five times with 0.2% digitonin or RIPA buffer (as above). Some
samples were subjected to in vitro kinase reactions with [32P]ATP,
quenched with 1% Triton X-100 buffer (as above), and secondarily immunoprecipitated with anti-Fyn mAb-coupled agarose as previously described
(46). Phosphoamino acid analysis was performed as previously
described (54).
The Journal of Immunology
therefore, this is not proof that these interactions cannot occur
in vivo.
To identify additional candidate proteins that interact efficiently
with the Fyn N-terminal domain, we screened a cDNA library
from the K46 B cell lymphoma using this domain as bait in the
two-hybrid system. The majority of yeast colonies that scored positive in this screen contained the full length tctex-1 cDNA (34),
which encodes a protein component of the ATP-dependent dynein
motor complex (37). The interaction with Fyn was specific, as
demonstrated in Figure 1, since the tctex-1 gene product did not
interact with the N-terminal domains of LynA, LynB, Blk, or
p56lck (Lck; see Fig. 5) as measured by activation of either His3
(histidine-free growth selection) or lacZ (b-galactosidase; data not
shown) reporters. In addition, the N-terminal domain of Fyn was
not observed to form homotypic interactions (Fig. 1). Tctex-1
failed to interact with Ig-a or Ig-b cytoplasmic domains in this
assay (data not shown), and we could not demonstrate any capacity
of Tctex-1 to “couple” Ig-a to Fyn when expressed as a third
protein in yeast two-hybrid experiments (data not shown). The
interaction of Fyn with Tctex-1 is clearly a strong protein-protein
interaction when compared with the lack of detectable interactions
between Fyn and Ig-a or Ig-b.
lacking. As shown in Figure 2, Northern blot analysis demonstrated ample expression of tctex-1 mRNA in all tissues examined
from BDF1 mice. Thymic expression was more pronounced than
that of spleen, while significant expression of tctex-1 mRNA was
observed in both B and T lymphocyte lines (Fig. 2). Less abundant
expression of tctex-1 was also evident in the brain, suggesting that
the protein is also available for interaction with Fyn in neuronal
cells (Fig. 2).
An affinity-purified pAb against recombinant Tctex-1 was prepared to test lymphocyte cell lines for protein expression. To characterize this pAb preparation, a C-terminal FLAG epitope-tagged
version of Tctex-1 was stably expressed in COS-7 cells. As shown
in Figure 3a, immunoprecipitation from a 35S metabolically labeled transfectant (clone C20) with either anti-Tctex-1 or antiFLAG Abs resulted in the purification of a specific band of about
15 kDa. This apparent mass is slightly higher than that predicted
for Tctex-1 (;14 kDa) due to the epitope tag. This band was the
major specific protein immunoprecipitated by the anti-Tctex-1
pAb preparation when compared with control immunoprecipitations (anti-human MHC class I and anti-tubulin mAbs in lanes 3
and 4 of Fig. 3a). In addition, the pAb preparation and anti-FLAG
mAb reproducibly immunoprecipitated similar amounts of
Tctex-1.
Several murine B and T cell lines were tested for Tctex-1 expression using the pAb preparation. Although Tctex-1 was difficult
to detect by immunoblotting with the pAb in whole cell lysates of
lymphocyte lines (one to two million cell equivalents per lane; data
not shown), it could be readily immunoprecipitated with this Ab
preparation from several lymphocyte cell lines and detected by
immunoblotting with the same Ab. The protein was observed in
immunoprecipitates from 50 to 70 million cell equivalents of the
fyn-transfected T cell hybridoma, N17 (10), and the B cell lymphomas K46, A20, and WEHI 231.7 (Fig. 3b; data not shown). The
specific reactive band was observed at 14 kDa, as predicted, and
this migration corresponded exactly to that of recombinant Tctex-1
as shown in Figure 3b. No significant improvement in the amount
of Tctex-1 immunoprecipited from lysates of K46 cells could be
demonstrated upon lysis with the addition of other nonionic detergents, harsher RIPA buffer, 10 mM ATP, or nocodazole pretreatment of cells, and only slight improvement was achieved using a monoclonal anti-dynein intermediate chain Ab (data not
shown). Therefore, both mRNA and the protein product of the
tctex-1 gene were detectably expressed in hemopoietic tissues as
well as T and B lymphocyte lines, but protein levels were routinely
low in these cells.
Despite significant efforts, we could not detectably coimmunoprecipitate the two proteins from Fyn-transfectants of COS-7 cells,
NIH 3T3 fibroblasts, or the T cell hybridoma N17 by immunoprecipitation of either protein or dynein intermediate chain (data not
shown). Detergents tested in these studies included Triton X-100,
digitonin, CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1propanesulfonate), and RIPA, and detection methods employed
were either 35S metabolic labeling, immunoblotting, or in vitro
phosphorylation of immunoprecipitated kinase in the presence of
[32P]ATP. These results suggest that the protein interaction is either transient in these cells or the off rate of the interaction is high,
thereby limiting detection after washing of immunoprecipitates.
Expression of Tctex-1 in lymphoid cells
The tctex-1 gene had been cloned from sperm cDNA and identified
as a product of the t complex of mice (34). Previous analysis by
Northern blotting had determined that the gene was strongly expressed in the testes and ovaries (34) and weakly in the thymus of
wild-type mice, but a detailed analysis in hemopoietic tissues was
Fyn interaction with Tctex-1-GST fusion protein in vitro
Due to the difficulties in demonstrating coimmunoprecipitation, we
tested the capacity of recombinant Tctex-1-GST fusion protein to
interact with Fyn in cell lysates. Digitonin lysates of Fyn-transfected NIH 3T3 cells (45) were adsorbed with various GST fusion
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FIGURE 2. Tctex-1 mRNA is expressed in all tissues and immune cell
lines tested. Northern blot of tctex-1 expression in various tissues and lymphocyte lines. Total RNA (10 mg/lane) was sequentially hybridized with
the full length tctex-1 cDNA (top) and b-actin cDNA (bottom). The various
murine cell lines tested are representative of proB cells (38B9), immature
B cells (WEHI-231.7), mature B cells (K46), and mature T cells (CTLL-2).
1731
1732
Fyn INTERACTS WITH THE DYNEIN LIGHT CHAIN, Tctex-1
proteins, including Tctex-1, and precipitates were washed and subjected to in vitro [32P]ATP phosphorylation reactions to detect
associated phosphoproteins. As presented in Figure 4a, GST fusion proteins of the cytoplasmic domains of Ig-a, and to a lesser
extent, Ig-b, were significantly phosphorylated in this assay, while
Tctex-1-GST fusion protein was only weakly phosphorylated. The
Tctex-1-GST fusion protein, however, selectively coprecipitated a
phosphoprotein band of about 59 kDa, which comigrated with
p59fyn. Reimmunoprecipitation from these phosphorylation reactions with an anti-Fyn mAb confirmed that the kinase had associated with the Tctex-1-GST fusion protein (Fig. 4a). The [32P]p59
band from Figure 4a exclusively contained phosphotyrosine,
which further verified its identity as autophosphorylated Fyn (Fig.
4b). Although Tctex-1-GST did not provide a good substrate for
the adsorbed Fyn in this assay, the minimal 32P labeling of Tctex1-GST was also found to be exclusively incorporated onto tyrosine
residues (Fig. 4b). These results suggest that Tctex-1 can be
weakly tyrosine phosphorylated by Fyn kinase. Finally, Fyn binding to Tctex-1-GST was confirmed by immunoblotting with
anti-Fyn Ab as demonstrated in Figure 4c. The Ig-a-GST fusion
protein was also able to adsorb Fyn in both of these assays as has
previously been reported (3), while Ig-b, PDGFR-KI, and the Nterminal domain of Fyn did not bind appreciable amounts of the
enzyme. In summary, the Tctex-1-GST fusion protein can selectively bind Fyn from digitonin lysates in vitro, thereby confirming
the yeast two-hybrid interaction.
Truncation analysis defining interacting sequences in Fyn and
Tctex-1
To identify the specific interacting domains in both Fyn and
Tctex-1, truncation analysis studies were undertaken in the yeast
two-hybrid system. When truncations of the 85-amino acid N-terminal domain of Fyn were tested as demonstrated in Figure 5a,
elimination of just the first 6 amino acids from the N terminus
completely abrogated the interaction. The C-terminal amino acids
of this domain, however, did not appear to contribute to the interaction with Tctex-1, yet amino acids 1 through 19 still exhibited
strong interaction. On the other hand, further truncation to amino
acids 1 through 10 completely abolished the interaction (see Fig.
5a). Previous studies by Timson Gauen et al. (32) have indicated
that amino acids 1 through 10, and in particular the lysine residues
at positions 7 and 9 of Fyn (Fig. 5b), are critical elements in the
interaction of this domain with ITAM-containing sequences on
lymphocyte Ag receptors. We also tested for involvement of lysines 7 and 9 in interaction with Tctex-1 by mutating them to
alanines in the complete N-terminal domain. As shown in Figure
5c, this mutated fragment of Fyn did not interact with Tctex-1,
indicating that these lysine residues are involved in interactions of
the kinase with both ITAM-containing sequences and Tctex-1.
Thus, Tctex-1 interacts with the first 19 amino acids of Fyn; lysine
residues at positions 7 and 9 are critical elements in this interaction
domain, although they are clearly not the only binding residues, as
determined in the truncation analysis (Fig. 5a).
Tctex-1 was also truncated and tested for interaction with Fyn in
the two-hybrid system. As shown in Figure 6, Tctex-1 was very
sensitive to truncation from both ends, since truncation of amino
acids 105 through 113 completely abolished the interaction with
Fyn, and elimination of amino acids 1 through 12 significantly
reduced the interaction. These results demonstrate that the protein
requires integrity of both termini to form the Fyn-interacting structural domain.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
FIGURE 3. Analysis of Tctex-1 protein expression in lymphocytes. a, Affinity-purified anti-Tctex-1 pAb specifically immunoprecipitates FLAG epitopetagged Tctex-1. COS-7 cells were stably transfected with a C-terminal FLAG-tagged Tctex-1 expression plasmid (clone C20), metabolically labeled with
[35S]cysteine/methionine, lysed in 1% digitonin, and immunoprecipitated with anti-FLAG mAb covalently conjugated to Sepharose beads (15 mg/30 ml of
beads) or GammaBindPlus Sepharose beads (30 ml) precoupled with anti-Tctex-1 pAb (3 mg), anti-human MHC class I mAb (W6/32, labeled huMHC-I;
75 ml of culture supernatant), or anti-tubulin mAb (3 mg). Immunoprecipitated proteins were separated on 17% SDS-PAGE, transferred to Immobilon-P
membrane, and detected by autoradiography. b, Immunoprecipitation of Tctex-1 from lymphocyte lines. The K46 (55 million/sample) and A20 B cell
lymphomas and the N17 T cell hybridoma (70 million each per sample) were lysed in 1% digitonin and immunoprecipitated with rabbit anti-mouse IgG
pAb, affinity-purified anti-Tctex-1 pAb (each 10 mg/sample), or preimmune serum from the same rabbit (10 ml), which were precoupled to 30 ml of
GammaBindPlus Sepharose beads. Immunoprecipitates were separated on 17% reducing SDS-PAGE, transferred to Immobilon-P membranes, and immunoblotted with anti-Tctex-1 pAb (10 mg/ml) and 125I-labeled protein A. The 16-kDa m.w. marker (lysozyme) routinely cross-reacted with anti-Tctex-1 pAb
as evident in the “marker” lane.
The Journal of Immunology
FIGURE 5. Truncation mapping of the domain within the N terminus of
Fyn that interacts with Tctex-1. a, The first 19 amino acids of Fyn interact
with Tctex-1. Truncation mutants of the N terminus of Fyn were tested as
Gal4 DB domain fusions for interaction with full length Tctex-1 fused to
Gal4 TA domain. Tctex-1 fused to the Gal4 DB domain was also tested for
interaction with the N-terminal domain of Lck kinase. Transfectants were
tested for activation of a histidine-free growth reporter and a b-galactosidase reporter. Magnitudes of reporter activation are indicated (11 5
strong, 2 5 none detected). b, Sequence of amino acids 1 through 19 of
murine Fyn that interacts with Tctex-1. Lysines 7 and 9, myristoylated
glycine 2 and palmitoylated cysteines 3 and 6 are marked. c, Mutation of
lysines 7 and 9 of the N terminus (-NH) of Fyn abrogates interaction with
Tctex-1. Mutant Fyn N-terminal domain (amino acids 1– 85) with lysines
7 and 9 changed to alanines (KK/AA) was compared with wild-type Fyn
domain (amino acids 1– 85) for interaction with Tctex-1 or the N-terminal
domain of Lck in the histidine-free growth assay.
Intracellular colocalization of Tctex-1 and Fyn proteins in
T lymphocytes during cytokinesis
Confocal immunofluorescence analysis was performed to determine the intracellular localization of Tctex-1. Analysis of
C-FLAG-Tctex-1-transfected COS-7 cells demonstrated that antiFLAG mAb (Fig. 7A) and anti-Tctex-1 pAb (identical pattern; data
not shown) diffusely stained the cytoplasm with the majority of
staining concentrated in the perinuclear region. Alternatively, microtubules (stained with anti-b-tubulin) emanated from the perinuclear Tctex-1 stained region and extended to the periphery of the
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FIGURE 4. Fyn kinase binds to a GST-fusion protein of Tctex-1 in
vitro. a, Fyn selectively binds to Tctex-1-GST fusion protein in vitro. Digitonin lysates of fyn-transfected fibroblasts (ZIP-Fyn cells; 2.5 3 106/sample) were adsorbed with the indicated GST fusion proteins or anti-Fyn
mAb and subjected to in vitro kinase reactions. Ten percent was retained
to analyze primary precipitations (left panel), and the remainder was reimmunoprecipitated with anti-Fyn mAb (right panel). Fusion proteins were
GST fused to cytoplasmic domains of Ig-a or Ig-b, kinase insert region of
platelet-derived growth factor receptor (PDGFR KI), full length Tctex-1, or
amino acids 1 through 85 of Fyn (Fyn-NH). Phosphoprotein bands corresponding to the Tctex-1-GST fusion protein (open arrow) and the p59fyn
band reimmunoprecipitated from the Tctex-1-GST precipitation (closed
arrow) are marked. b, GST-Tctex-1 and associated p59fyn are exclusively
phosphorylated on tyrosine residues. The marked 32P-labeled phosphoprotein bands in panel a were subjected to phosphoamino acid analysis. c,
Anti-Fyn imunoblotting confirmed Fyn binding to Tctex-1-GST. Digitonin
lysates of ZIP-Fyn cells (14 3 106/sample) were adsorbed with GST fusion
proteins or anti-Fyn mAb. SDS-PAGE-separated samples were immunoblotted with anti-Fyn pAb.
1733
1734
Fyn INTERACTS WITH THE DYNEIN LIGHT CHAIN, Tctex-1
Tctex-1 at the center of the cleavage furrow in the cell crosssections as presented in Figure 7C. Overall, this stage of cell division was the only interval during which we consistently observed
intracellular colocalization of Fyn with Tctex-1. Tctex-1 staining
was always perinuclear throughout all other stages of the cell cycle, and we never observed staining of the nucleus or plasma membrane. In conclusion, we observed highly reproducible colocalization of Fyn and Tctex-1 during cytokinesis at both the cleavage
furrow and mitotic spindles in this T cell hybridoma.
Discussion
cells (Fig. 7A). Tctex-1 staining was not detected in the nucleus or
plasma membrane.
Double-staining intracellular immunofluorescence studies were
also performed to determine whether Tctex-1 and Fyn are colocalized in lymphocytes. The fyn-transfected murine T cell hybridoma, N17 (10), provided detectable levels of kinase for these studies. Elevated Fyn expression in N17 cells was clearly evident in
immunoblots of whole cell lysates and immunofluorescent staining
when compared with other lymphocyte cell lines (data not shown).
Most N17 cells exhibited Fyn staining that was predominantly
concentrated to the plasma membrane, although some cells exhibited significant foci of Fyn staining within the cytoplasm (Fig. 7B)
as previously reported by Ley et al. (23). Although Tctex-1 staining was concentrated within the perinuclear/cytoplasmic region
and generally distinct from Fyn-stained regions, a subset (;25%)
of the T hybridoma cells demonstrated distinct cytoplasmic foci of
Fyn, some of which were clearly colocalized with Tctex-1 (Fig.
7B). We surmised that this inconsistent colocalization of Fyn with
Tctex-1 within the population of T hybridoma cells might represent colocalization only at a distinct stage(s) of the cell cycle. To
subdivide the cells into distinct stages of cycle, the N17 cells were
sorted by DNA content on FACS into G0/G1, S, and G2/M stages
before intracellular Ab staining. Although no consistent colocalization was identified in the G0/G1 or S stage populations (data not
shown), the G2/M stage population exhibited a unique overlap in
staining that was observed in all dividing cells. As shown in Figure
7C, this population is enriched in cells undergoing cytokinesis,
which exhibited a striking colocalization of Fyn and Tctex-1 at the
developing cleavage furrow that forms as daughter cells begin to
divide. In addition, colocalization was evident in these cells at the
mitotic spindles. It is interesting to note that Tctex-1, but not Fyn
or b-tubulin, seems to interdigitate through the segregated chromosomes and connects the mitotic spindles with the cleavage furrow via fibrous arrays. b-Tubulin staining, although a major component of the mitotic spindles, did not colocalize with Fyn and
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FIGURE 6. Truncation mapping of Tctex-1 domains that interact with
Fyn. The N-terminal domain of Fyn (amino acids 1– 85) was tested as a
Gal4 TA fusion protein for interaction capacity with the truncation mutants
of Tctex-1 fused to the Gal4 DB domain as assessed by growth on histidine-free medium. Magnitudes of reporter activation are indicated (11 5
strong, 1 5 weak, 2 5 none detected).
We have identified and characterized a novel protein interaction
between the N-terminal domain of Fyn protein tyrosine kinase and
Tctex-1, which has recently been described as a light chain component of the cytoplasmic ATP-dependent dynein motor complex.
The interaction was confirmed in vitro using a Tctex-1-GST fusion
protein. Tctex-1 can interact with the first 19 amino acids of Fyn,
and lysines at positions 7 and 9 of this sequence are critical elements for the interaction. Immunofluorescent confocal microscopy
showed that the two proteins consistently colocalize during cytokinesis at both the cleavage furrow and mitotic spindles of a T cell
hybridoma, suggesting a role for this interaction in cell division.
The ubiquitous expression of Tctex-1 suggests that its interaction
with Fyn might also occur in other tissues in which Fyn is readily
expressed, most notably neuronal tissues. In view of the limited
proliferation of neuronal tissues, the interaction in such tissues
would be less likely to play a role in the division of mature cells
than in lymphocytes, suggesting additional functional roles for the
interaction. Kai et al. (56) have also recently reported tctex-1 as
one of several cDNAs that were cloned using a larger portion of
Fyn as a bait in the yeast two-hybrid system, although they did not
attempt to address the interaction biochemically or further define
the interaction domains.
Amino acids 1 through 19 of Fyn (see Fig. 5b), which were
identified as interacting with Tctex-1, encompass the domain that
interacts with unphosphorylated ITAM motifs in lymphocyte Ag
receptors (SH4, amino acids 1–10 (31, 32)) and contain three lipid
modification sites (Fig. 5b). Cotranslational myristoylation of the
glycine at position 2 is believed to be a permanent alteration, while
posttranslational palmitoylation of cysteines at positions 3 and 6 is
a reversible modification (24 –29, 57). Interestingly, the two lysines at positions 7 and 9 within the Fyn interactive sequence are
critical elements for interactions of Fyn with both Tctex-1 (Fig. 5c)
and ITAM sequences (32). These basic residues have previously
been implicated in enhancing plasma membrane association of Src
family kinases by interacting with negatively charged phospholipid head groups (24). Our results and those of Timson Gauen et
al. (32) indicate that these lysine residues and presumably other
residues in the extreme N-terminal domain of Fyn are also important for protein-protein interactions. One attractive hypothesis
would be that a lymphocyte activation or cell cycle-related event
might promote depalmitoylation of Fyn and thereby release it from
the membrane, exposing the domain that interacts with Tctex-1. It
is unlikely that the Fyn fusion proteins are palmitoylated in our
yeast experiments, since palmitoylation of this sequence is considered to be dependent upon nearby myristoylation in Src family
kinases (24, 26, 28), which is not possible with the initiating methionine of Fyn fused to Gal4. Mutation of the two lysines of Fyn
has been shown to alter its localization from the plasma membrane
to the cytoplasm (32), although our mutational studies indicate that
this localization is not due to Tctex-1 binding (Fig. 5c).
The colocalization of Fyn with Tctex-1 during cytokinesis is a
particularly intriguing result in light of other recently published
The Journal of Immunology
1735
FIGURE 7. Intracellular localization of Tctex-1 and colocalization with
Fyn during cytokinesis. A, COS-7 cells were transfected with C-FLAGTctex-1 and intracellular staining was performed with anti-FLAG Ab to
visualize Tctex-1 (FITC; green) and b-tubulin (Texas Red). B, Fyn and
Tctex-1 colocalize in a subpopulation of N17 T cell hybridomas. The N17
cell line was double-stained with anti-Tctex-1 pAb (Oregon Green 488)
and anti-Fyn mAb (Texas Red). The majority of cells demonstrate segre-
gated plasma membrane staining of Fyn and perinuclear staining of Tctex-1, and colocalization of the two proteins was evident only in a subpopulation of cells as visualized in yellow (filled arrowheads), while some
cells contained distinct cytoplasmic foci of Fyn at sites that are not enriched in Tctex-1 (red; outlined arrowheads). C, Fyn and Tctex-1 colocalize in the cleavage furrow and at the mitotic spindles of N17 T cell hybridomas undergoing cytokinesis. N17 cells in G2/M phase of the cell cycle
were sorted by FACS and double-stained with either Oregon Green-labeled
anti-Tctex-1 pAb or anti-Fyn pAb plus Texas Red-labeled anti-b-tubulin
mAb or anti-Fyn mAb. Each horizontal series shows separate green and red
channels of the indicated staining, and both channels merged in the same
focal slice from individual cells. The bottom three cells demonstrate colocalization (yellow in the merged image) of Tctex-1 and Fyn at the cleavage furrow (open arrowheads) and mitotic spindles (closed arrowheads).
These cells are characteristic of anaphase to early telophase, since they are
all beginning to pinch into separate daughter cells. All cells found at this
stage of cytokinesis exhibited this representative colocalization. The bar in
each panel denotes 10 mm.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
reports. Of particular interest is a study by Yasunaga et al. (22),
which determined that proB cells cultured from fyn-deficient mice
grew essentially normally until transferred to defined serum-free
conditions, at which point these cells arrested during cytokinesis at
telophase. In striking contrast, proB cells from normal animals
continued through the cell cycle in these serum-free conditions.
Their results indicate that Fyn plays a critical role in cell division,
although its requirement can be overcome by serum-derived
growth factors that presumably bypass the Fyn deficiency block.
The same report also demonstrated the localization of Fyn in the
cleavage furrow at anaphase of normal proB cells, which we have
reproduced in our studies of a T cell hybridoma. Taken together
with this genetic evidence, our results suggest that Tctex-1 might
provide the crucial scaffold link that tethers Fyn to cytoskeletal
motor structures in lymphoid cells, where it functions during cytokinesis. Previous observations of decreased proliferative capacity of thymocytes from fyn-deficient mice (11, 12) might, in fact,
be partially explained by this requirement and further reinforces
the importance of this newly identified function for the kinase.
Ley et al. (23) have reported that Fyn is almost exclusively
localized at the centrosome and mitotic spindles of interphase and
mitotic T cells, respectively. Since dynein mediates retrograde
transport toward these structures, the association of Fyn with
Tctex-1 in the dynein complex could clearly mediate this localization of Fyn in lymphocytes. Although we have observed predominantly plasma membrane localization of Fyn in interphase
cells using two different Abs in the murine T cell hybridoma, N17
(and other T lymphomas; data not shown), a subpopulation of cells
demonstrated distinct cytoplasmic foci of Fyn staining, some of
which colocalized with Tctex-1 (Fig. 7C). Ley et al. used a polyclonal Ab directed to the C-terminal domain of Fyn (23), while our
Abs were N-terminal reactive, which may account for the predominantly plasma membrane staining pattern in our studies. Roche et
al. (58) have demonstrated a requirement for Src kinases, including
Fyn, at an earlier stage of the mitotic cell cycle. They reported
increased activity of Fyn and other Src kinases in G2/M phaseblocked fibroblasts. In addition, fibroblasts were arrested before
prophase, in the same report, by microinjection of an anti-Fyn/Src/
Yes Ab (the same Ab used by Ley et al.) or a GST fusion protein
of the Fyn SH2 domain. Finally, Marie-Cardine et al. (59) reported
that T cell activation results in tyrosine phosphorylation of a-tubulin and that this phosphorylated a-tubulin can bind the Fyn SH2
domain. Katagiri et al. (60) have also noted tyrosine phosphorylation of tubulin during monocyte differentiation of HL-60 cells
and concomitant association of Fyn and Lyn with tubulin. These
1736
Acknowledgments
We thank Drs. Pierre Cosson, Marco Colonna, Ulrich Deuschle, Ed
Palmer, Jeff Bluestone, Jean Pieters, and Salvatore Valitutti for valuable
advice and comments on the manuscript. We also thank Drs. David Beach,
John Cambier, Pierre Chevray, Noemi Fusaki, José Garcı́a-Sanz, Beat Imhof, Marina Cella, Andrius Kazlauskas, Daniel Nathans, Antonius Rolink,
Tohru Tezuka, Takeshi Watanabe, and Tadashi Yamamoto for generously
providing reagents; Bea Pfeiffer and Hans Spalinger for photography; and
Irina Serbodova for generating several plasmid constructs for these studies.
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results taken together suggest potentially important roles for Fyn
recruitment to and phosphorylation of microtubule cytoskeletal elements during cellular activation and mitosis and suggest that Tctex-1 might be an important mediator of the Fyn recruitment.
What is the role of this interaction during the cell cycle? Although we can only speculate, colocalization of Fyn with Tctex-1
at the cleavage furrow/mitotic spindles and fyn requirements for
cytokinesis in proB cells (22) suggest specific roles for these proteins in the division of lymphocytes. Alternatively, the binding of
Fyn to Tctex-1 may occur as a capture mechanism for specific
cargo in dynein-mediated protein sorting during mitosis. Our understanding of the complex molecular events occurring at the
cleavage furrow is only currently unfolding. The role of actin filaments and associated structures in this process is clear, but microtubules and even dynein appear also to play roles (38, 61).
Cytoplasmic dynein has also previously been shown to play roles
in centrosome separation, anaphase B spindle elongation, and positioning of mitotic spindles during mitosis (38 – 40). Recruitment
of Fyn to dynein by Tctex-1 might affect some or all of these
mitotic events.
Although Tctex-1 does not seem to serve as a major substrate
for Fyn in our in vitro phosphorylation studies (Fig. 4a and direct
mixing in in vitro phosphorylation reactions, data not shown), it
was nevertheless detectably tyrosine phosphorylated. Other proteins within the dynein complex may, however, be more efficacious substrates once the kinase is recruited. Ley et al. (23) have
described protein tyrosine phosphorylation surrounding the Fyn at
microtubule organizing centers, and as previously mentioned,
a-tubulin is a potential tyrosine phosphorylated substrate for Fyn
at this location (59, 60). Phosphorylation events identified to date
during cytokinesis have predominantly focused on the myosin
chains, which undergo serine/threonine phosphorylation (reviewed
in Ref. 61). Components of the cytoplasmic dynein complex have
been reported to be phosphorylated during cell cycle and transport
processes, but again, only serine and threonine phosphorylation
has been identified (47, 62– 64). Karki et al. (65) recently reported
the association of casein kinase II with dynein, which appears to
mediate some of this protein phosphorylation and thereby appears
to affect function of the motor complex. Determination of the functional roles of recruited Fyn during cytokinesis in lymphocytes and
the possibility of consequent tyrosine phosphorylation of the dynein complex and associated structures should allow for many enlightening future investigations.
In summary, accumulating evidence is implicating Fyn kinase as
an important effector within the cytoskeleton of lymphocytes, particularly during mitosis. The identification of a direct interaction of
the Fyn N-terminal domain with the cytoplasmic dynein motor
complex light chain, Tctex-1, provides a novel mechanism for the
recruitment of Fyn to this distinct intracellular location.
Note Added in Proof. Two potential Fyn substrates that might be
important during cytokinesis are the cleavage furrow-associated
protein PSTPIP (66) and the inositol 1,4,5-trisphosphate receptor
(67).
Fyn INTERACTS WITH THE DYNEIN LIGHT CHAIN, Tctex-1
The Journal of Immunology
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