(Received for publication, July 7, 1995; and in revised form, September 13, 1995)
From the
Several cellular signal transduction pathways activated by
middle-T in polyomavirus-transformed cells are required for viral
oncogenicity. Here we focus on the role of phosphatidylinositol
3-kinase (PI 3-kinase) and Ras and address the question how these
signaling molecules cooperate during cell cycle activation. Ras
activation is mediated through association with SHC GRB2
SOS
and leads to increased activity of several members of the
mitogen-activated protein (MAP) kinase family, while activation of PI
3-kinase results in the generation of D3-phosphorylated
phosphatidylinositides whose downstream targets remain elusive. PI
3-kinase activation might also ensue as a direct consequence of Ras
activation. Oncogenicity of middle-T requires stimulation of both Ras-
and PI 3-kinase-dependent pathways. Mutants of middle-T incapable to
bind either SHC
GRB2
SOS or PI 3-kinase are not oncogenic.
Sustained activation and nuclear localization of one of the MAP
kinases, ERK1, was observed in wild type but not in mutant
middle-T-expressing cells. Wortmannin, an inhibitor of PI 3-kinase,
prevented MAP kinase activation and nuclear localization in
middle-T-transformed cells. PI 3-kinase activity was also required for
activation of the MAP kinase pathway in normal serum-stimulated cells,
generalizing the concept that signaling through MAP kinases requires
not only Ras- but also PI 3-kinase-mediated signals.
Proteins expressed early in the virus life cycle of
polyomavirus, the tumor antigens (T antigens), are responsible for
tumor formation in virus-infected animals and virus-mediated
transformation of cells in culture(1) . Large tumor antigen
(large-T) is a nuclear protein known to immortalize primary cells in
culture (2) while middle tumor antigen (middle-T) causes
phenotypic changes associated with malignant cell growth(3) .
The activity of middle-T results from its association with
intracellular signal-transducing proteins like members of the Src
family of tyrosine kinases (c-Src, Fyn, and c-Yes)(4) , the 85-
and 110-kDa subunits of a phosphatidylinositol 3-kinase (PI
3-kinase)()(5) , the catalytic and regulatory
subunits of protein phosphatase 2A (PP2A)(6, 7) , and
the SH2 domain-containing protein SHC (8, 9) whose
putative role is to activate the Ras signaling
pathway(10, 11) . More recently, middle-T
immunoprecipitates have been found to contain a member of the 14-3-3
family of proteins, some of which are involved in stimulating
ADP-ribosylation(12) .
Middle-T activates intracellular
signal transduction pathways mediated by PI 3-kinase and Ras,
respectively. The latter becomes activated upon association of the
SHCGRB2
SOS complex with middle-T. Middle-T-transformed
cells show an increase in the fraction of the GTP-bound form of Ras (13) and transfection with genes suppressing Ras activity
results in reversion to a more normal phenotype(14) . Activated
Ras stimulates cell growth and differentiation through a kinase cascade
involving Raf and MEK culminating in the activation and nuclear
translocation of several members of the MAP kinase family (15, 16, 17) . Middle-T has also been shown
to activate transcription factors of the AP1 family like Jun and Fos (13, 18) or Myc(19) , the former being direct
targets of MAP
kinases(20, 21, 22, 23, 24) .
The ability to activate transcription of cellular genes through MAP
kinases is a prerequisite for cell transformation and delineating the
underlying mechanisms is therefore of pivotal importance in
understanding virus-mediated cell transformation.
In this study we
investigate the signals activated by middle-T through
SHCGRB2
SOS and PI 3-kinase, respectively, and evaluate
their importance for T antigen oncogenicity. Activation and
translocation of ERK1 to the nucleus was observed in cells expressing
wild type (WT) middle-T. Cells expressing T antigen mutants unable to
bind SHC and PI 3-kinase, respectively, showed neither activation nor
nuclear translocation of MAP kinases, suggesting that both pathways
feed into the MAP kinase cascade. Similarly, we found that PI 3-kinase
was also required for MAP kinase activation and translocation in
untransformed cells stimulated with growth factors.
The
reporter plasmids pGl2muPA-8.2 and pGl2muPA-35 were constructed by
inserting the murine uPA gene promoter (from -8.2 kilobase pairs
to +398 base pairs with respect to the transcription initiation
site) upstream of the luciferase-coding region of the promoterless
plasmid pGL2-basic (Promega). In p3xAP1-tk-luc, ()three
consensus AP1 elements were inserted upstream of the minimal promoter
of the tymidine kinase gene (-46 to +52) containing the TATA box
and the transcription initiation site, which is linked to the
luciferase gene. The control plasmid, pGl2-control (Promega) contains
the SV40 enhancer and promoter. pfos-luc contains a human c-fos promotor (-711 to +45) linked to the luciferase
gene(27) .
pSRAmSOS1 contains the coding region of a
dominant negative form of SOS under the control of the human T-cell
lymphotrophic virus (type I)-long terminal repeat
promoter(28) . The plasmid pCMV N
raf encodes a dominant
negative form of Raf under the control of the cytomegalovirus
promotor(29) .
Figure 1: Time course of ERK1 and ERK2 activation. Control NIH 3T3 cells (A) and stable cell lines expressing either wild type (B) or 1178T middle-T (C) or Y250F middle-T (D) were serum-starved for 40 h followed by stimulation with 10% calf serum. ERK1 and ERK2 activity were determined using myelin basic protein as substrate at various time points after serum stimulation. For each time course, one representative experiment is shown. Closed circles, data for ERK1; open circles, data for ERK2. The absolute values of the maximum activities for the various cell lines tested did not differ significantly.
Figure 2: ERK activity in control and middle-T-transformed cells. A, middle-T-expressing eEnd2 endothelioma cells(26) , NIH 3T3 control cells, and mT7 cells were analyzed for ERK1 and ERK2 kinase activity using myelin basic protein as substrate. Numbers indicate activity measured in a PhosphorImager. B, Western blot of control NIH 3T3 cells (lanes 1-3), cells transformed with WT middle-T (lanes 4-6), 1178T middle-T (lanes 7-9), or Y250F middle-T (lanes 10-12). Lanes 1, 4, 7, and 10 represent samples from asynchronously growing cells; lanes 2, 5, 8, and 11 from serum-starved cells; and lanes 3, 6, 9, and 12 from cells stimulated for 15 min with 10% calf serum. C, ERK1/ERK2 Western blot of NIH 3T3 cells stimulated for 15 min in the absence (lane 3) and presence (lane 4) of 100 nM wortmannin. Lane 1, asynchronously growing cells; lane 2, arrested cells.
Figure 3: Activation of promoter activity by middle-T. A, NIH 3T3 cells were transiently transfected with the indicated reporter plasmids alone (open bars) or together with 0.05 µg of pcDNAmT expression plasmid (closed bars). MT7 cells stably expressing middle-T (hatched bars) were transfected with the indicated reporter plasmid. Promoter activity was determined as described under ``Experimental Procedures.'' One representative experiment is shown. B, NIH 3T3 cells were transiently transfected with 1 µg of the reporter plasmid muPA-8.2 together with pcDNA1, pcDNAmT, pcDNAmT1178T, pcDNAmTY250F, pcDNAmT1387, pcDNANG59, or pcDNAdl1015. Dominant negative SOS- (cross-hatched bars), dominant negative Raf- (hatched bars), or MKP-1 (open bars) encoding expression plasmids were cotransfected with the middle-T vectors. Promoter activity was determined as described under ``Experimental Procedures.'' One representative experiment is shown.
So far we have shown that middle-T activates MAP kinases. Earlier reports suggest that constitutively activated MEK, the kinase acting upstream of ERK1 and ERK2, is sufficient for cell transformation (34, 35, 36) . This implies that Ras-mediated signaling is sufficient for ERK activation. An analysis of various middle-T mutants, on the other hand, suggests that both SHC- and PI 3-kinase-initiated pathways are required for T antigen oncogenicity(4, 37) . To address this discrepancy, we investigated a series of non-oncogenic middle-T mutants (Table 1). 1178T (31) shows dramatically reduced binding of PI 3-kinase, while Y250F (8, 9, 32) is deficient in associating with SHC and thereby unable to initiate signaling through Ras. NG59 (38) binds none of the molecules characterized so far, 1387T (a truncated mutant protein lacking the membrane anchor sequence) only binds PP2A(39, 60) , and dl1015 associates with all cellular enzymes described so far, but is unable to activate PI 3-kinase(40, 41) . Mutant genes were introduced into NIH 3T3 cells and activation of MAP kinase measured in the luciferase reporter assay. Fig. 3B shows that only WT middle-T fully stimulated the reporter gene. NG59, 1387T, and dl1015 were almost completely inactive, while 1178T and Y250F were about 50% as effective as WT in inducing the uPA promoter. Residual stimulation of the uPA promoter by mutant middle-Ts was totally blocked by dominant negative SOS and Raf or by MKP-1. These findings further support the view that both SHC- and PI 3-kinase-initiated signaling pathways are required for efficient stimulation of the MAP kinase cascade.
Figure 4: Translocation of ERK1 to the nucleus in middle-T-expressing cells. Figure shows asynchronously growing NIH 3T3 cells (A), and cells starved for 40 h and immunostained for ERK1 before (B) and 1 h after addition of 10% calf serum (C). Panel D, ERK1 immunostaining of asynchronously growing middle-T-expressing cells; panel E, serum-starved middle-T-expressing cells; panel F, serum-stimulated cells; panel G, ERK1 immunostaining; panel H, middle-T-specific staining of REF-52 cells microinjected with pcDNAmT plasmid; panel I, ERK1 staining; panel K, middle-T staining of REF-52 cells microinjected with pcDNAY250FmT plasmid; panel L, shows ERK1 immunostaining of synchronized F111 cells 1 h after serum stimulation; panel M, as in L but stimulated in the presence of 100 nM wortmannin.
Figure 5: Translocation of ERK1 to the nucleus in middle-T-expressing cells. Starved REF-52 cells were microinjected with pcDNAmT, pcDNA1178TmT, pcDNAY250FmT, or pcDNAdl1015mT expression plasmids. After injection the cells were kept for 8 h in low serum and immunostained for both middle-T and ERK1. The gray bars indicate the percentage of middle-T-expressing cells that show nuclear localization of ERK1. As a control the same number of uninjected control cells was counted. The same experiment was performed in the presence of 100 nM wortmannin (black bars). In each experiment several hundred cells were counted.
To test the relevance of these findings for growth factor-mediated
signaling in normal cells, we studied the localization of ERK1 in
serum-stimulated fibroblasts in the absence and presence of wortmannin (Fig. 4, L and M). Nuclear accumulation of
ERK1 was completely blocked by the drug, in agreement with the data
shown for microinjected cells expressing middle-T. Similarly, the M shift indicative of activation of ERK1 and ERK2
was abolished when G
-arrested cells were stimulated with
serum growth factors (Fig. 2C).
We also investigated the effect of PI 3-kinase on mitogen-stimulated induction of S phase in mouse NIH 3T3 and F111 rat fibroblasts. Table 2shows that wortmannin blocked serum-induced initiation of DNA synthesis measured as bromodeoxyuridine incorporation into cellular DNA, while control cells efficiently entered S phase.
Figure 6: Focus formation on F111 fibroblasts by various middle-T mutants. F111 rat fibroblasts were transfected with 20 µg of the middle-T-encoding expression plasmids pcDNAmT, pcDNAY250FmT, and pcDNA1178TmT. For double transfections, 10 µg of each expression plasmid were used. Focus assays were performed as described under ``Experimental Procedures.'' The data shown represent the average of four independent experiments.
Middle-T transforms cells through association with a variety
of proteins involved in cell signaling (4, 37) and
activates intracellular signal transduction pathways mediated by the PI
3-lipid kinase and Ras, respectively. It is well established that Ras
stimulates the MAP kinase
pathway(15, 16, 17) . Owing to its ability to
induce the phosphorylation of cellular transcription factors like Jun
and Fos, middle-T has been suggested to activate the MAP kinase
cascade(13, 18, 46) . It has also been shown
that middle-T activates genes coding for various transcription factors (19, 47) like Fos (18) and Jun(46) .
Investigating the effect of middle-T on the MAP kinase pathway we
studied several parameters. (i) We measured the activity of MAP kinases in vitro using myelin basic protein as substrate; (ii) we
determined the shift in the apparent M of MAP
kinases upon cell stimulation indicative of activation upon
phosphorylation by MEK; (iii) we measured the activity of MAP
kinase-regulated promoters in reporter plasmid-transfected cells using
a luciferase reporter gene; and (iv) we studied the intracellular
localization of a representative member of the MAP kinase family, ERK1,
by immunofluorescence microscopy.
Expression of WT middle-T, but not
of transformation-defective mutant proteins, resulted in high basal MAP
kinase activity in asynchronously growing or serum-starved cells. WT
middle-T-expressing cells showed increased ERK1 and ERK2 activity, as
determined in the myelin basic protein phosphorylation assay but showed
no corresponding shift in the apparent M of these
kinases typical for MAP kinase activation in growth factor-stimulated
cells. This might be explained by the fact that middle-T-transformed
cells do not accumulate in G
upon serum starvation and are
refractory to further stimulation by growth factors. The M
shift in MAP kinases might only arise in cells
entering the cell cycle from G
but not in cycling cells.
Alternatively, the M
shift of ERK1 and ERK2 might
be transient preceding translocation to the nucleus. Since MAP kinases
are constitutively localized in the nucleus of middle-T-transformed
cells, transient phosphorylation by MEK might escape detection on
Western blots. Earlier data obtained with cells overexpressing mutant
forms of ERK1 and ERK2 suggest that the shift in M
resulting from phosphorylation by MEK as well as enzymatic
activity of ERK1 and ERK2 are not required for translocation to the
nucleus(15, 17, 43) . A change in activity
and/or specificity of PP2A upon association with T antigens might
further contribute to altered phosphorylation and activity of ERK1 and
ERK2 in polyomavirus-transformed cells. In agreement with this idea,
recently published papers show that SV40 small-T activates the MAP
kinase pathway by blocking PP2A-mediated
down-regulation(48, 49) .
A detailed analysis of the pathways targeted by middle-T was performed in cells co-transfected with middle-T and a reporter gene consisting of the coding region derived from the firefly luciferase gene under the control of the uPA, Fos, or AP1 promoters, respectively. Emphasis was on the uPA promoter, since it has been shown previously that expression of uPA is increased in middle-T-induced endotheliomas. This protease has been shown to be one of the major determinants in T antigen-induced morphological transformation(26) . Signaling through the MAP kinase pathway was dramatically reduced in cells expressing transformation-defective middle-T mutants. Dominant negative Raf, dominant negative SOS, and overexpression of MKP-1, a phosphatase involved in down-regulation of MAP kinases, blocked T antigen-mediated activation of the uPA promoter reminiscent of experiments performed earlier with tyrosine kinase growth factor receptors(33, 50) .
While short term
treatment of cells with growth factors is sufficient to transiently
activate MAP kinases, sustained activation accompanied by nuclear
translocation of ERK1 and ERK2 are required for mitogenic stimulation
of cells through this pathway(17, 51, 52) . A
variety of T antigen mutants was used to address the question which of
the pathways initiated by middle-T were required for sustained
activation and nuclear translocation of MAP kinases. Our data show that
only WT middle-T efficiently stimulates nuclear translocation of ERK1
establishing that SHC-mediated activation of Ras was not sufficient for
activation of the MAP kinase cascade. Thus PI 3-kinase activation seems
to be an important factor in T antigen-mediated MAP kinase activation
and mitogenic signaling. To corroborate these findings, we treated
middle-T-expressing cells with wortmannin, an inhibitor of PI 3-kinase.
Relocalization of ERK1 upon middle-T expression was completely blocked
by the drug, confirming the results obtained with middle-T mutants
unable to activate PI 3-kinase. Nuclear translocation of MAP kinase
observed in a small fraction of 1178T middle-T-expressing cells is
therefore most likely the consequence of residual PI 3-kinase activity
and not due to a PI 3-kinase-independent
pathway(5, 31, 53) . This explanation is
consistent with earlier studies showing that mutation of tyrosine 315,
the major binding site for the 85-kDa subunit of PI 3-kinase, to
phenylalanine in the 1178T mutant, reduced but did not completely
abolish oncogenicity and PI 3-kinase activity(5, 31) .
Remaining activity might be the consequence of residual binding of p85
to this mutant protein. Alternatively, elevated D3-phosphorylated
PIP levels might arise from SHC-mediated Ras activation,
resulting in stimulation of PI 3-kinase(54) . Our
interpretation of these data was confirmed with another mutant, dl1015,
still able to associate with this enzyme yet unable to activate PI
3-kinase activity(41) .
The fact that Y250F middle-T was totally defective in causing nuclear localization of ERK1 and ERK2 yet only 2-fold reduced in inducing the uPA gene can be explained in three ways. (i) Detection of nuclear localization of ERK1 by immunostaining depends on accumulation of a significant fraction of the enzyme in the nucleus while only a small amount of nuclear ERK1 might be sufficient to activate the uPA promoter; (ii) Transient transfections of Y250F middle-T together with the reporter plasmid had to be performed in the presence of serum that might compensate for the defect of this mutant in initiating the Ras pathway; (iii) activation of the uPA promoter might also ensue after phosphorylation of transcription factors in the cytoplasm followed by their translocation to the nucleus. Oncogenicity of WT middle-T is most likely the result of the induction of a complex set of cellular genes upon phosphorylation of various transcription factors by MAP kinases. Our data suggest that activation and translocation of MAP kinases to the nucleus upon expression of middle-T best correlates with mitogenicity and oncogenicity of this protein. Partially defective mutants unable to cause relocalization of MAP kinases do not initiate the cell cycle, suggesting that some of the crucial substrates of MAP kinases are nuclear.
Wortmannin blocked nuclear translocation of MAP kinases in middle-T-expressing cells and efficiently prevented phosphorylation and relocalization of these kinases to the nucleus in serum-stimulated control cells, establishing our findings as a general phenomenon during mitogenic stimulation of cells. Support for our observation also comes from the fact that wortmannin has been shown by others to reduce the efficiency of signaling through the MAP kinase pathway upon insulin or serum treatment(55, 56) .
To test the hypothesis that
SHCGRB2
SOS- and PI 3-kinase-initiated pathways operate
independently on MAP kinases, we performed focus assays with cells
transfected with two plasmids encoding 1178T and Y250F middle-T,
respectively. While each mutant alone was unable to induce foci, a
combination of both efficiently transformed cells indicating that the
two pathways can be initiated from separate middle-T complexes and
efficiently cooperate to activate the MAP kinase cascade.
In
summary, we have shown here that both SHCGRB2
SOS as well as
PI 3-kinase-induced signaling pathways are required for full
stimulation of the MAP kinase cascade by polyomavirus middle-T or serum
growth factors. It remains the goal of further studies to identify the
level at which these pathways crosstalk. Recently published data
demonstrate that a constitutively activated PI 3-kinase activates Ras,
Raf, and MAP kinases and stimulates transcription of Fos, suggesting a
role for this enzyme upstream of Ras(57) . Studies with mutant
growth factor receptors point to a role of PI 3-kinase in mitogenesis,
cell migration, and receptor internalization(58) . Whether
localization of MAP kinases is affected by mutations preventing binding
of PI 3-kinase to activated growth factor receptors remains to be
determined. Other signaling molecules such as S6 kinase and the
proto-oncogene akt1 have been identified as putative
downstream targets of PI 3-kinase(59) . It will be interesting
to investigate whether these kinases are necessary for
middle-T-mediated transformation and nuclear translocation of MAP
kinases.