(Received for publication, October 10, 1996, and in revised form, December 3, 1996)
From the Gene Medicine Department, Rhône-Poulenc Rorer, 13 Quai Jules Guesde, 94403 Vitry sur Seine, Cedex, France
Sam68 is the main tyrosine-phosphorylated and
Src-associated protein in mitotic cells. Sam68 exhibits a conserved
functional KH (hnRNPK homology) RNA binding domain and binds single
strand nucleic acids. Tyrosine phosphorylation mediates the interaction of Sam68 with many SH3- and SH2-containing proteins and negatively regulates its nucleic acid binding properties. But the function and the
impact of Sam68 on cell signaling and cell proliferation remains
elusive. We report here the identification of a natural isoform of
Sam68 with a deletion within the KH domain. This isoform, called
Sam68KH, is specifically expressed at growth arrest upon confluency
in normal cells. In cells that do not enter quiescence at confluency
such as Src-transformed cells, no recruitment of Sam68
KH is
observed. Transfected Sam68
KH inhibits serum-induced DNA synthesis
and cyclin D1 expression. Sam68 overcomes these effects, suggesting
that isoforms of Sam68 are involved, through KH domain signaling, in
cell proliferation, and more precisely in G1/S
transition.
Sam68 is the main tyrosine-phosphorylated and Src-associated
protein in mitotic cells (1-5). Based on several experimental evidences, Sam68 turned out to be encoded by the hump62
cDNA previously described as the cDNA of the GAP-p62 associated
protein (5). Sam68 exhibits a conserved functional KH (hnRNPK
Homology) RNA binding domain and RGG boxes, two landmarks of RNA
binding proteins (6, 7), and was indeed shown to bind single strand nucleic acids (8, 9). Because Sam68 is tyrosine-phosphorylated in
mitotic cells and has five proline-rich domains, Sam68 interacts both
through SH2- and SH3-mediated interactions not only with the Src family
tyrosine kinases but also with signaling molecules such as Grb2 and
phospholipase C-1 (10, 11). Sam68 is therefore envisioned as a
multifunctional SH3 and SH2 domain binding protein that could link Src
family kinases to putative downstream proteic and nucleic
acid effectors. A physiological function or an impact of Sam68 on
signaling events remains to be elucidated.
Binding of Sam68, Sam68KH, and Sam68 (203-443) to Poly(U)
Wild type Sam68(1-443), Sam68KH(
170-208), and
Sam68(203-443) were translated in vitro (Promega).
Sam68(203-443) was obtained by PCR1 using
specific primers that introduced an in-frame initiation codon. The
translated proteins were incubated 30mn at 4 °C in presence of
poly(U) beads in binding buffer (Tris-HCl, pH7, 5, 10 mM;
MgCl2, 2.5 mM; Triton, 0.5%; NaCl, 100 mM). Beads were washed three times in binding buffer, and
bound proteins were eluted in Laemmli buffer for 10 min at 95 °C.
Specificity was assessed by competition with an excess of soluble
poly(U). The poly(U) binding activity (binding percent) of each
polypeptide is expressed as percentage of specific binding relative to
the total binding. The results represent the average of four
independent experiments.
Tissue Expression of Sam68 and Sam68KH
5 µg of total RNA (Clontech) were
reversed transcribed, and 10% of the synthetized cDNAs were used
for amplification of Sam68 and Sam68KH cDNAs with the following
5
primers: AATTTTGTGGGGAAGATTCTTGGA, specific for Sam68, encompassing
nucleotides 511-534 of Sam68 cDNA and located within the KH
domain, and CCTGTCAAGCAGTATCCCAAGGAG, specific for Sam68
KH, being
located across the new splice junction and corresponding to nucleotides
486-510 of Sam68
KH cDNA (nucleotides 486-627 of Sam68
cDNA). The specificity of this primer is due to the fact that only
the nucleotidic sequence of Sam68
KH is entirely colinear with the
sequence of the primer. The same 3
primer (GTATGTATCATCATATCCATATTC)
was used for both Sam68 and Sam68
KH cDNAs amplification and
corresponds to nucleotides 1101-1125 of Sam68 cDNA. Primers
specificities were checked to avoid nonspecific cross-reactions. The
Sam68 and Sam68
KH cDNAs at concentrations ranging from 100 to
0.1 pg were also amplified by PCR as standards. A 600-base pair
fragment was amplified in all samples. The figure depicts 10% of the
PCR products separated on a 1.5% agarose gel. As a control, the
amounts of cDNA used in each experiment were evaluated by
amplifying the cDNA encoding
actin with specific oligonucleotides (12).
Multiple tissue Western blot (Clontech) was incubated
with antibodies according to the manufacturer's instructions. The
anti-Sam68 monoclonal antibody is commercially available (P20120;
Transduction Laboratories), and the anti-Sam68KH antibodies were
purified from the serum of rabbit injected with a peptide corresponding to the Sam68
KH-specific sequence:
(H)-Cys-Lys-Gln-Tyr-Pro-Lys-Glu-Glu-Glu-Leu-Arg-Lys-(OH). Antibody
specificity was checked on recombinant Sam68 and Sam68
KH proteins
expressed in Sf9 using a baculovirus expression system (BaculoGold,
Pharmingen). This expression system allowed to express both Sam68 and
Sam68
KH recombinant proteins as 10% of the total proteins. 10 µg
of infected Sf9 lysates were used as control of the specificities of
the antibodies. Revelations were performed with Enhanced
ChemiLuminescence (Amersham Corp.).
105 NIH3T3 cells and105 NIH3T3 cells transformed by c-Src (Y567F) were synchronized by 24 h of serum starvation and then stimulated by 10% of fetal calf serum up to 48 h.
Cells were washed twice in PBS, and total cell lysates were prepared by
scrapping in Laemmli buffer with 6 M urea. For each sample,
10 µg of proteins were analyzed on 10% SDS-PAGE. Antibodies against
Sam68 and Sam68KH were as described above.
1.5105 NIH3T3 cells were transfected
with myc-tagged Sam68 (1 or 5 µg) or myc-tagged
Sam68KH (1 or 5 µg) SV40 expression vectors. Transfection
experiments were performed using Lipofectamine (Life Technologies,
Inc.) and peptide H1 under conditions that ensured the transfection of
60% of the cells, as controlled by
-galactosidase staining. In a
first set of experiments, cells were rinced twice in PBS 24 h
after transfection and synchronized by serum starvation during 24 additional hours. Growth was then stimulated by 10% fetal calf serum
during 12 h. BrdUrd incorporation was then analyzed both by
immunoassay (5-bromo-2-deoxy-uridine labeling and detection kit III,
Boehringer Mannheim) or FACS analysis. In this case, BrdUrd (30 µM) was added 30 min before collection. Cells were stained for DNA content with propidium iodide and for DNA synthesis with a fluorescein-conjugated anti-BrdUrd antibody. The results represent the average of five independent experiments for immunoassay detection, and three for FACS analysis. The 100% value was arbitrary assigned to the BrdUrd incorporation observed after serum stimulation of quiescent cells. In a second set of experiments, transfected cells
were asynchronously grown, and S phase entry was analyzed 24 h
after transfection as described above. In these experiments, protein
expressions were evaluated by direct Western blot analysis of 10 µg
of cell extracts (4-20% gradient SDS-PAGE). Total lysates were
obtained as described previously. Proteins were detected with the 9E10
anti-myc monoclonal antibody or a cyclin D1 monoclonal antibody (HD11, Santa Cruz).
While using RT-PCR to clone Sam68 cDNA from human placental
RNA, we identified a cDNA fragment identical to Sam68 sequence except for a deletion within the KH domain. This in-frame deletion spanned the region encoding amino acids residues 170-to 208 of Sam68
(Fig. 1a). Three positive clones were
isolated after screening of 106 recombinant phages from a
human gt11 placenta cDNA library with a 24-mer oligonucleotide
specific for the deletion. Two clones encompassed an entire open
reading frame that was identical to that of Sam68 with the exception of
the deletion of the KH RNA binding domain (Fig. 1a). We
called this open reading frame Sam68
KH for deleted form of Sam68.
These isoforms probably arise from alternative splicing of a single
pre-mRNA species, because the analysis of their remaining common
sequences appeared identical, even in the 5
and 3
noncoding
regions.
Both isoforms as well as the carboxyl-terminal of Sam68 which does not
contain any known RNA binding motif (8), were in vitro
translated. The same amount of each protein was incubated with
poly(U)-agarose beads (9, 13), and specifically bound proteins were
quantified. Sam68KH bound poly(U) one-fifth as much as did Sam68
(Fig. 1b). However, when compared with the carboxyl-terminal part of Sam68, which was used as a negative control, it appeared that
Sam68
KH retained some impaired RNA binding properties. This could be
due to the presence of RGG boxes in the NH2-terminal domain
of Sam68 that are still present in the deleted isoform and indicates
that the deletion of the KH domain does not alter the overall structure
of the protein.
Because the predicted sizes of both mRNAs differ by only 117 bases
and because Northern blot analysis of several human adult tissue
samples revealed only one mRNA species of 2.9 kilobases,2 we performed RT-PCR to evaluate
the expression of Sam68 and Sam68KH mRNAs. Sam68
KH mRNA
was detected in the different adult tissues (Fig.
2a), and to be able to distinguish between
the expression of the two isoforms in human samples, we generated
antipeptide antibodies that would only recognize Sam68
KH. These
antibodies recognized a single band in the different human samples,
intensified in skeletal muscle and in liver, in agreement with the
RT-PCR data (Fig. 2b). This band is likely to represent
endogenous Sam68
KH protein because the antibodies recognized
recombinant Sam68
KH but not Sam68 expressed using baculoviruses. In
addition, this band was not detected when the immunoblots where
realized in the presence of 50 µg/ml of competing peptide
antigen2 (Fig. 2b).
Using an anti-Sam68 monoclonal antibody directed against the KH domain,
which failed to recognize Sam68KH (Fig. 2b), Sam68 was
significantly detected in every sample, although it appeared less
represented in brain, skeletal muscle, and liver. These data indicated
that Sam68
KH is a natural isoform of Sam68 and that the ratio
between these two isoforms varies among different adult tissues. These
variations could reflect the involvement of Sam68
KH in
differentiation and/or proliferative processes, which could depend on
functional differences between the two isoforms.
Sam68KH has a crippled KH domain and therefore impaired RNA binding
properties. It can be envisionned that the two isoforms could have
distinct and possibly antagonistic functions. Although no biological
role has been assigned yet to Sam68, its specific phosphorylation and
Src association are restricted to mitosis and support the hypothesis
that Sam68 could be involved in the regulation of cell
proliferation.
In order to examine whether or not Sam68 and Sam68KH expressions
could vary according to cell growth, we performed Western blot
experiments on NIH 3T3 cells under different conditions of cell
proliferation. Synchronized cells were analyzed as they reached confluency (Fig. 3a). In quiescent cells
synchronized by serum starvation for 24 h as well as in
proliferating cells, only Sam68 expression was observed. Quiescence and
cell cycle progression of the cells were checked by FACS analysis,
which showed that BrdUrd incorporation was detected 12 h after
serum stimulation, indicating the progression of the cells in phase S. 24 h after serum stimulation, both Sam68 and Sam68
KH were
detected. In cells stimulated by serum for 36 and 48 h, which
became confluent, no BrdUrd incorporation was observed, indicating the
quiescence of the cells. This correlated with a marked increased in the
expression of Sam68
KH. These data indicate that Sam68
KH is
expressed at confluency in NIH3T3 cells, suggesting that this protein
could be recruited upon growth arrest. Oncogenic transformation bypass this growth arrest upon confluency in fibroblasts. We performed the
similar set of experiments on NIH3T3 cells transformed by c-Src (Y527F)
(Fig. 3b). Under the same conditions of culture, the cells
continued to incorporate BrdUrd and Sam68
KH was not detected. These
data indicate that Sam68
KH is observed upon confluency only in cells
that enter quiescence in these cell culture conditions.
We next addressed whether or not Sam68KH was able per se to inhibit
cell proliferation and specifically S phase entry in response to serum
stimulation (14). Transfection of plasmid encoding
myc-tagged Sam68 did not modify the BrdUrd incorporation of
quiescent NIH3T3 cells stimulated by serum and transfected with empty
vector (Fig. 4a). In sharp contrast,
expression of myc-tagged Sam68
KH led to more than 50%
inhibition of BrdUrd incorporation. A cotransfection of Sam68
expression vector plasmid was able to rescue the inhibition induced by
Sam68
KH. The fact that Sam68 had no effect per se in the absence of
exogenous Sam68
KH suggest that Sam68 is not rate-limiting in NIH3T3
cells. In primary rat embryo fibroblasts, however, exogenous
transfected Sam68 increased by 30% the BrdUrd
incorporation.2 The same transfection experiments were
realized in exponentially growing NIH3T3 cells and also indicated that
Sam68
KH inhibited BrdUrd incorporation and that Sam68 rescued this
inhibition induced by its isoform (Fig. 4b), the expression
of the recombinant Sam68 and Sam68
KH being controlled by Western
blot analysis using the anti-myc 9E10 antibody (Fig.
4c). Because cyclin D1 is rate-limiting for G1
to S phase entry in fibroblasts (15, 16), we performed Western blot
analysis on the exponentially growing NIH3T3 cells using an anti-cyclin
D1 monoclonal antibody in order to further characterize the
antagonistic impact of the transfected Sam68 and Sam68
KH. Our data
indicate that the inhibition induced by Sam68
KH and the rescue by
Sam68 are respectively correlated with a down-regulation and a recovery
of cyclin D1 expression (Fig. 4c).
These results are the first data that document a function for Sam68 and
support the hypothesis that Sam68 participates in the control of cell
proliferation by promoting the S phase entry. This function of Sam68
appears to involve its KH domain and probably its RNA binding
properties. It has been shown that these RNA binding properties are
impaired when Sam68 is phosphorylated (9). Because Sam68 is
tyrosine-phosphorylated and Src-associated only in mitosis, it is
likely that Sam68 impact on G1/S transition does not rely directly on its association with Src. However, because Src is involved
both in G1 (17-19) and in G2 (20) and is also
activated in mitosis (21, 22), we cannot exclude that Sam68 functions in cell cycle progression depend, directly or indirectly, on Src activity. Our results suggest also that two isoforms of Sam68 compete
for common partners to regulate cell growth, although we cannot exclude
that they have also specific protein or nucleic targets. Future
experiments will try to identify the RNA targets of Sam68 that seem
critical for S phase entry and will lead toward the clarification of
the role of Sam68 in Src signaling. Sam68KH will prove useful to
study Src pathways and cellular differentiation, a paradigm of cell
arrest.
We thank Michel Maratrat for FACS analysis, Isabelle Delumeau for stimulating discussions, and Florence Risbec for excellent technical assistance.