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Cell Growth & Differentiation Vol. 11, 279-292, June 2000
© 2000 American Association for Cancer Research

Oncogenic Transformation of Cells by a Conditionally Active Form of the Protein Kinase Akt/PKB1

Amer M. Mirza, Aimee D. Kohn, Richard A. Roth and Martin McMahon2

Cancer Research Institute, University of California, San Francisco/Mt. Zion Cancer Center, San Francisco, California 94115 [A. M. M., M. M.], and Department of Molecular Pharmacology, Stanford University, Palo Alto, California 94305 [A. D. K., R. A. R.]


   Abstract
TOP
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The Akt/PKB protein kinase is implicated in the control of cell cycle progression and the suppression of apoptosis in cancer cells. Here we describe the use of a conditionally active form of Akt/PKB (M+Akt:ER*) to study the ability of this protein to influence biological processes that are central to the process of oncogenic transformation of mammalian cells. Activation of M+Akt:ER* in Rat1 cells elicited alterations in cell morphology and promoted anchorage-independent growth in agarose with high efficiency. Consistent with these observations, activation of M+Akt:ER* suppressed the apoptosis of Rat1 cells that occurs after the detachment of these cells from extracellular matrix. Furthermore, activation of M+Akt:ER* was sufficient to promote the progression of quiescent Rat1 cells into the S and G2-M phases of the cell cycle. In accord with this is the observation that activation of M+Akt:ER* led to decreased expression of the cyclin-dependent kinase inhibitor p27Kip1 with a concomitant increase in cyclin-dependent kinase-2 activity. Perhaps surprisingly, activation of M+Akt:ER* or expression of a constitutively active form of Akt led to rapid activation of MAP/ERK Kinase (MEK) and the extracellular signal-regulated kinase (ERK)/mitogen-activated protein (MAP) kinases in Rat1 cells. However, pharmacological inhibition of MEK by PD098059 did not inhibit the morphological alterations of Rat1 cells that occur after M+Akt:ER* activation. These data suggest that M+Akt:ER* can activate a number of pathways in Rat1 cells, leading to significant alterations in a number of biological processes. The conditional transformation system described here will allow further elucidation of the ability of Akt to contribute to both the normal response of cells to mitogenic stimulation and the aberrant proliferation observed in cancer cells.


   Introduction
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 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 References
 
Akt was identified, by homology to the catalytic {alpha} subunit of the cyclic AMP-dependent protein kinase (1) , as a putative serine/threonine kinase RAC{alpha} (related to cyclic AMP-dependent PKA3 and PKC; Ref. 2 ). Because of the similarity to both PKA and PKC, it has also been referred to as PKB{alpha} (3) . c-Akt (4) was independently identified as the cellular homologue of v-Akt (5) , the oncogene of the AKT-8 murine leukemia virus (6) . The AKT-8 retrovirus was originally isolated from a spontaneous thymoma in an AKR mouse and was subsequently shown to be capable of transforming mink lung fibroblasts in culture (7) . After inoculation of susceptible mouse strains, this virus produced thymic lymphomas (6) , and cells expressing v-Akt were found to be highly oncogenic when injected into nude BALB/c mice (8) . To date, three mammalian isoforms of c-Akt have been identified (2 , 5 , 9 , 10) . Each is composed of three distinct and conserved domains: an NH2-terminal PH domain; a catalytic protein kinase domain; and a short COOH-terminal regulatory region related to a similar region of PKC. The Akt isoforms differ with respect to their distribution in various tissues as well as the levels of expression of each isoform, although the significance of these differences remains unclear.

c-Akt is a serine/threonine kinase (11, 12, 13) that is rapidly activated in response to a variety of cytokines and growth factors (11 , 12 , 14 , 15) . The current model for the activation of c-Akt suggests that growth factor-mediated activation of PI3'-kinase leads to increased production of PI3'-lipids. Such lipids bind to the PH domain of c-Akt, leading to its recruitment to the plasma membrane and alleviating the repressive influence of the PH domain on the kinase domain. Once at the plasma membrane, it is acted upon by one or more upstream kinases that are themselves regulated by phospholipid products of PI3'-kinase (16 , 17) . A phosphoinositide 3,4,5-trisphosphate-dependent protein kinase 1 has been shown to phosphorylate c-Akt at Thr-308, thereby increasing its activity >30-fold (18) . In 293 cells, rapid induction of c-Akt activity by insulin and insulin-like growth factor-I is dependent upon phosphorylation of two amino acids, Thr-308 and Ser-473 (16 , 19) . The identity of the Ser-473 kinase remains elusive.

The PI3'-kinase/Akt pathway is evolutionarily conserved and has been reported to play a key role in the insulin signal transduction pathway in Caenorhabditis elegans, where it appears to be critical for the inhibition of dauer arrest (20) . In mammalian cells, Akt is believed to be responsible for the insulin-induced inhibition of glycogen synthase kinase-3 and the subsequent activation of glycogen synthase (21, 22, 23) , glucose uptake, and glucose transporter-4 translocation (22 , 24) . Furthermore, Akt appears to play a role in insulin-induced protein synthesis by activation of eukaryotic initiation factor 2 (25) , as well as phosphorylation and inhibition of 4E-BP1, a repressor of mRNA translation (26) . In addition to its role in metabolism, Akt plays a role in the regulation of apoptosis in metazoan organisms from Drosophila (27) to mammals (28, 29, 30) . It also appears to be involved in the growth factor-mediated survival of neurons. Experiments with the PC12 pheochromocytoma cell line (31) , which is dependent upon PI3'-kinase for its survival (32) , and with H19-7 hippocampal neuronal cells demonstrate that pharmacological inhibitors of PI3'-kinase, as well as dominant-negative forms of Akt, inhibit the activation of Akt and induce apoptosis (33) . The mechanism of Akt-mediated suppression of apoptosis is unclear. However, it has been suggested that this may occur through Akt-induced phosphorylation of the proapoptotic proteins BAD, caspase-9, and FKHRL1 (34, 35, 36) . Furthermore, both Fas- and Myc-induced apoptosis (37 , 38) are abrogated after activation of PI3'-kinase and Akt (39 , 40) .

Of interest is the fact that Akt is a proto-oncogene (4) , and Akt isoforms have been shown to be overexpressed in the MCF7 breast cancer cell line (9) . Moreover, it has been found to be amplified >20-fold in a gastric adenoma (5) , and Akt2 is overexpressed in a significant number of ovarian (41) and pancreatic (42) cancers. In addition, Akt appears to be a downstream target for transforming oncogenes such as Ras and v-Src (43) . However, it is not clear how Akt participates in oncogenesis.

To explore the ability of Akt to promote oncogenic transformation, we have established a transformation system in Rat1 fibroblasts using a stably expressed, conditionally active form of Akt (M+Akt:ER*; Ref. 44 ). In brief, M+Akt:ER* was constructed by fusing the c-Src myristylation targeting sequence to a constitutively active form of Akt lacking the PH domain (M+Akt). Conditionality was conferred in a manner similar to that described for other protein kinases (45 , 46) by fusing M+Akt to a modified form of the hormone binding domain of the mouse ER (ER*) that binds 4-HT but is refractory to estrogen (47) . As a result, M+Akt:ER* is rapidly activated in response to 4-HT and elicits effects that have been attributed to endogenous cellular Akt (24 , 44) . In this study, we demonstrate that activation of M+Akt:ER* induces oncogenic transformation in Rat1 fibroblasts, as manifested by alterations in cell morphology and the capacity to form colonies in agarose. In addition, activation of M+Akt:ER* was sufficient to induce cell transformation by its dual ability to suppress apoptosis and to promote the entry and progression of cells through the cell cycle. The latter correlated with a sustained decrease in levels of the cdk inhibitor p27Kip1 and an increase in the activity of cdk2. These data are consistent with previous reports that PI3'-kinase and the PTEN PI3'-lipid phosphatase participate in the regulation of cell cycle progression and apoptosis in human cancer cells. Thus, we are able to directly implicate Akt in the regulation of events ascribed previously to the PTEN/PI3'-kinase as a whole. We also observed that activation of M+Akt:ER* led to rapid activation of the p42/p44 ERK/MAP kinases; however, ERK/MAP kinase activation was not required for the morphological alterations in Rat1 cells. These data indicate that Akt has the capacity to regulate key biological processes that participate in oncogenic transformation in mammalian cells and are consistent with a role for Akt in human tumorigenesis.


   Results
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 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 References
 
Akt Activation Induces Cellular Transformation of Rat1 Fibroblasts.
To determine the effects of Akt activation on cellular transformation, we used retrovirus-mediated gene transfer to generate pools of Rat1 cells expressing a membrane-targeted, conditionally active Akt/PKB (M+Akt:ER*). As a control, we expressed a myristylation-defective form of the protein (M-Akt:ER*), which is not membrane associated and therefore nontransforming. The M+Akt:ER* construct is reported to elicit many of the downstream events attributed to wild-type or constitutively active forms of Akt, whereas M-Akt:ER* does not (24) . Western blot analysis revealed that both M-Akt:ER* and M+Akt:ER* were expressed equally well in Rat1 cells (data not shown). Although the ability of Akt to induce changes in cell morphology in chicken fibroblasts has been reported (48) , here we confirm and extend these observations using M+Akt:ER* in mammalian cells. The ability of Akt to induce cellular transformation was assessed in three ways: by its ability to induce changes in cell morphology; by its ability to elicit an altered colony morphology; and by its ability to promote cell growth in an anchorage-independent manner in agarose.

To determine whether the activation of M+Akt:ER* was able to induce changes in cell morphology, confluent cultures of Rat1 fibroblasts expressing M+Akt:ER* (Rat1::M+Akt:ER*) were cultured in the absence or presence of 100 nM 4-HT. Representative data from one of seven experiments are shown. Within 24 h, cells cultured in the presence of 4-HT were found to be refractile by phase-contrast microscopy. After 48 h (Fig. 1B)Citation , the cells exhibited an elongated, refractile, spindle-like morphology. In areas, these cells began to overgrow one another, indicating that they had lost the ability to be contact inhibited. In contrast, cells cultured in the absence of 4-HT (Fig. 1A)Citation grew to confluence in an even monolayer and displayed normal contact inhibition.



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  		Fig. 1. Akt activation is sufficient to induce cellular transformation of Rat1 fibroblasts. Confluent monolayers of Rat1::M+Akt:ER* cells were split 1:3 and plated in the absence (A) or the presence (B) of 100 nM 4-HT. M+Akt:ER*-induced morphological transformation in Rat1 fibroblasts is dependent upon membrane localization and 4-HT. Cells infected with either a myristylation-defective (M-Akt:ER*; C) or a myristylation-competent (M+Akt:ER*) form of M+Akt:ER* (D) were cultured to confluence and treated with 100 nM 4-HT for an additional 18 h. E, M+Akt:ER* induced an altered colony morphology on plastic. Cells (1 x 104) were plated in media in the absence or the presence of 100 nM 4-HT as indicated and cultured for 14 days, fixed with methanol, and Geimsa stained. Conditionally active forms of Akt (M+Akt:ER*) and Raf (EGFP{Delta}Raf-1: AR), as well as a constitutively active form of Akt (myrAkt), induced anchorage-independent growth. F, Rat1::M+Akt:ER* fibroblasts were plated in DMEM containing 0.35% agarose at a concentration of 1 x 103 and 1 x 104 cells/well in the absence or presence of 500 nM 4-HT as indicated. After 14 days in culture, cells were stained with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and photographed. G, similarly, Rat1 cells expressing myrAkt or Rat1::eGFP{Delta}Raf-1:AR fibroblasts were plated in agarose. EGFP{Delta}Raf-1:AR was activated by incubation with 300 nM testosterone.

 
It has been suggested that the recruitment of Akt to the membrane is essential for its activation and function (49) . To determine whether membrane localization of M+Akt:ER* is essential for its ability to induce morphological transformation, cells expressing either M+Akt:ER* (Fig. 1D)Citation or a myristylation-defective M-Akt:ER (Fig. 1C)Citation were grown to confluence and then cultured for 18 h in the presence of 100 nM 4-HT. Only cells expressing the membrane localized M+Akt:ER*, became morphologically altered, and lost the ability to be contact inhibited in response to 4-HT. These data indicate that membrane localization of M+Akt:ER* is likely to be essential for its full activation, targeting to relevant substrates and effectors, or both.

To ascertain whether activation of M+Akt:ER* altered the growth characteristics of Rat1::M+AKT:ER* fibroblasts on extracellular matrix, we compared the growth of cells plated at subconfluent densities (1 x 104 cells/10-cm plate) and cultured for 14 days (Fig. 1E)Citation . Representative data from one of four experiments are shown. Cells cultured in the absence of 4-HT formed flat, diffuse colonies that displayed contact inhibition. In contrast, cells that expressed an activated M+Akt:ER* formed dense, multilayered colonies of refractile cells. Because of their increased cell density, these colonies were revealed as more darkly staining macroscopic foci.

To determine whether the ability to elicit alterations in cell morphology was restricted to Rat1 cells, M+Akt:ER*expressing NIH3T3 and Rat1A fibroblasts cell lines were derived by retrovirus infection. Activation of M+Akt:ER* in these cells also induced focus formation at a high frequency; however, its effects on morphology changes in these cells were quite subtle and quite unlike the effects elicited by Ras or Raf (data not shown). Thus, the ability of activated M+Akt:ER* to elicit changes in colony morphology was not restricted to Rat1 fibroblasts.

To address whether the activation of M+Akt:ER* in Rat1 fibroblasts was capable of inducing anchorage-independent growth, the ability of these cells to form colonies in agarose was examined. Cells were plated in agarose at 103 or 104 cells/well in the absence or presence of 4-HT to activate M+Akt:ER*. After 7 days in culture, cells expressing M+Akt:ER* formed macroscopic colonies in the presence of 4-HT. After 14 days, the cultures were stained with the vital dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Fig. 1F)Citation . Measured over six independent experiments, the frequency of colony formation induced by activated M+Akt:ER* was 31.3 ± 3.8% and 0.84% ± 0.74% in the absence of 4-HT. The frequency of colony formation induced by activated M+Akt:ER* was similar to that induced by a constitutively active form of Akt (myrAkt). In a separate experiment, the ability of M+Akt:ER* to induce colony formation in Rat1 cells was compared with that of conditionally active EGFP{Delta}Raf-1:AR and was found to be comparable (Fig. 1G)Citation .

Rapid, Dose-dependent Activation of M+Akt:ER* by 4-HT.
To better understand the mechanism of M+Akt:ER* activation, the kinetics of its induction after the addition of 4-HT were determined. Confluent, serum-deprived Rat1::M+Akt:ER* fibroblasts were treated with 300 nM 4-HT to activate M+Akt:ER* for different periods of time, and its activity was assessed by immune-complex kinase assay (Fig. 2A)Citation . M+Akt:ER* was activated within 15 min after the addition of 4-HT, with maximum activation occurring between 1 and 2 h. Thereafter, the activity was sustained at peak levels for the duration of the time course.



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  		Fig. 2. Time course of M+Akt:ER* activation after treatment with 4-HT. A, Rat1::M+Akt:ER* cells were grown to confluence, cultured in the absence of serum for 4 h, and then treated with 300 nM 4-HT for various times from 5 min to 3 h or ethanol alone (solvent control) for 3 h, at which time cell lysates were prepared. M+Akt:ER* was immunoprecipitated with a polyclonal anti-ER ({alpha}ER) antibody. The activity of M+Akt:ER* was measured using an immune-complex kinase assay with histone 2B (H2B) as a substrate. The amount of M+Akt:ER* present in each kinase reaction was determined by Western blot analysis with the polyclonal anti-ER antibody. B, M+Akt:ER* activation is dependent upon the concentration of 4-HT. Rat1::M+Akt:ER* cells were cultured as described above and then stimulated with various concentrations of 4-HT from 3 to 1000 nM or with 20% FCS as indicated. An Akt immune-complex kinase assay was performed as described above with H2B as substrate (lower panel). The phosphorylation status of serine residue 473 was determined by Western blot analysis with an anti-phosphoserine 473 antibody (middle panel). The blot was subsequently stripped, and Western blot analysis with the polyclonal anti-ER antibody was used to determine the amount of M+Akt:ER* in each lane.

 
To determine effective doses for the stimulation of M+Akt:ER* kinase activity, confluent, serum-deprived Rat1::M+Akt:ER* fibroblasts were stimulated with various concentrations of 4-HT from 0 to 1000 nM for 1 h. M+Akt:ER* activity increased with the concentration of 4-HT added and was maximal between 30 and 100 nM 4-HT (Fig. 2B)Citation . Increases in the dose of 4-HT beyond 100 nM did not significantly increase the kinase activity of M+Akt:ER*. The results indicate that the kinase activity of M+Akt:ER* may be titrated by altering the concentration of 4-HT added to cell culture media.

It has been reported that Akt must be phosphorylated on both threonine residue 308 and serine residue 473 to be fully activated (16 , 19) . M+Akt:ER* became phosphorylated on Ser-473 upon stimulation with 4-HT. This phosphorylation was detectable at 4-HT concentrations of 100-1000 nM. In addition, treatment of Rat1::M+Akt:ER* cells with a high concentration of serum was also able to induce M+Akt:ER* phosphorylation and activity. These data are consistent with the notion that M+Akt:ER*, similar to wild-type Akt, may still in part be regulated by PI3'-lipids. However, the induction by serum was not as robust as with 4-HT. When given in combination, serum and 4-HT treatment led to hyperactivation of M+Akt:ER* kinase activity (data not shown). Taken together, these results indicate that M+Akt:ER* activity is rapidly induced in response to 4-HT and is titratable to levels higher than observed after acute stimulation with serum.

An apparent discrepancy in this experiment is the activation of M+Akt:ER*, as measured by kinase assays in the absence of detectable Ser-473 phosphorylation at 30 and 100 nM 4-HT. It is not clear whether this reflects an uncoupling of Ser-473 phosphorylation from M+Akt:ER* activation or a reflection of the sensitivity of the phospho-Ser-473-specific antiserum. The most likely explanation for the apparent discrepancy is that the Akt immune-complex kinase assay may simply be more sensitive than the phosphospecific antibody. At lower levels of Akt activity there may be a correspondingly low level of Ser-473 phosphorylation that is not detectable with the phospho-specific antibody.

Activation of M+Akt:ER* Protects Rat1 Cells from Apoptosis.
Akt is believed to transduce survival signals from a number of cytokine receptors as well as survival signals elicited by the binding of integrins to the extracellular matrix (28) . Thus, the prevention of apoptosis may play an important role in promoting the anchorage-independent growth of Rat1::M+Akt:ER* cells in agarose and thereby contribute to M+Akt:ER*-induced cellular transformation. Accordingly, the effect of M+Akt:ER* activation on apoptosis was determined after the removal of both serum and extracellular matrix survival signals. Apoptosis was measured by Annexin V-FITC and PI staining, and flow cytometry was used to quantitate the percentage of apoptotic cells in the culture. Representative data from one of four experiments are shown (Fig. 3)Citation . Attached Rat1::M+Akt:ER* fibroblasts cultured in the absence of serum for 18 h either with or without activated M+Akt:ER* were alive and nonapoptotic because they excluded PI and were negative for Annexin V staining (Fig. 3, left panelCitation ). These results indicated that Rat1 cells are protected from apoptosis, even in the absence of serum, as long as they remain attached to the cell culture dish. As a result, the activation of M+Akt:ER* had little or no effect on cell viability under these conditions. However, when cells were detached from the monolayer and cultured in suspension in the absence of serum, activation of M+Akt:ER* had a significant effect on cell viability (Fig. 3, right panelCitation ). Cells cultured in suspension in which M+Akt:ER* was inactive underwent programmed cell death with ~42% of cells becoming Annexin V positive in 12 h. In contrast, cells in which M+Akt:ER* was activated were, at least in part, rescued from apoptosis because only 25% of these cells were Annexin V positive. Therefore, in Rat1 fibroblasts, activation of M+Akt:ER* was able to inhibit apoptosis induced by removal of cells from extracellular matrix. A time course for the onset of apoptosis under these conditions was determined after the placement of these cells in suspension in the absence or presence of an activated M+Akt:ER*. Asynchronously growing cells were cultured in suspension in the absence of serum for 0, 6, 12, 18, and 24 h with or without 4-HT to activate M+Akt:ER* (Fig. 3E)Citation . PI staining and flow cytometry analysis of fragmented nuclei were used to determine the percentage of apoptotic cells in culture at the indicated times. Our results indicate that after 24 h, the percentage of apoptotic cells increased from <=10% to almost 40%. However, in the presence of activated M+Akt:ER*, by 24 h the percentage of apoptotic cells was <=17%. Therefore, in Rat1 fibroblasts, activation of M+Akt:ER* was able to delay the onset of apoptosis. These observations are consistent with the ability of M+Akt:ER* to promote anchorage-independent growth (Fig. 1F)Citation and suggest that the ability of activated M+Akt:ER* to induce Rat1 cells to grow in agarose may, at least in part, be dependent on its ability to prevent apoptosis in these cells. To elucidate the molecular mechanisms underlying the prevention of apoptosis in Rat1::M+Akt:ER* fibroblasts, the expression of candidate cell survival and proapoptotic gene products was examined by Western blot analysis after activation of M+Akt:ER*. There was no change in the expression of the antiapoptotic proteins Bcl-2 or Bcl-xL. Thus, our findings here confirm previous work suggesting that Akt does not alter Bcl-2 or Bcl-xL expression (50) . In addition, we found that there was no change in the expression of the proapoptotic protein Bax. It remains to be seen whether M+Akt:ER* phosphorylates to inactivate the proapoptotic proteins Bad, caspase-9, or FKHRL1 in these cells, as has been reported previously in other cells (34, 35, 36) .



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  		Fig. 3. M+Akt:ER* protects Rat1 cells from apoptosis. Left panel, Rat1::M+Akt:ER* cells were grown to confluence, left untreated (A) or treated with 100 nM 4-HT (C), and cultured for 18 h in the absence of serum, at which time they were detached and stained with Annexin V-FITC and PI. Right panel, confluent monolayers were trypsinized, left untreated (B) or treated with 100 nM 4-HT (D), and cultured in suspension in the absence of serum for 12 h. Flow cytometry was used to determine Annexin V and PI staining and to determine whether cells were undergoing apoptosis. Live cells are found in the lower left (LL) quadrant, cells in the early stages of apoptosis are found in the lower right (LR) quadrant, and cells late in the apoptotic program are found in the upper right (UR) quadrant. E, asynchronously growing Rat1::M+Akt:ER* cells were placed in suspension on ultra-low attachment plates in the absence of serum. PI staining and flow cytometry were used to score the percentage of apoptotic cells with fragmented nuclei both in the absence (-4-HT) or presence (+4-HT) of M+Akt:ER* activation. The time indicated represents the number of hours the cells were in suspension.

 
M+Akt:ER* Activation Is Sufficient to Induce DNA Synthesis and Cell Cycle Progression.
Increased cell cycle progression may also contribute to the growth of transformed Rat1::M+Akt:ER* fibroblasts. To determine the proliferative potential of these cells, clonal populations were established by selecting individual colonies growing in agarose. These SAR1 cells displayed rapid changes in cell morphology, becoming transformed within 24 h after activation of M+Akt:ER* (data not shown). The high frequency (>30%) with which the SAR1 cells form colonies in soft agar would suggest that these cells are representative of the population at large and may arise as a result of the levels of M+Akt:ER* expression rather than through the acquisition of additional transforming events. To elucidate the role, if any, of activated M+Akt:ER* in cell cycle progression and to determine whether activation of M+Akt:ER* in quiescent SAR1 fibroblasts was sufficient to induce DNA synthesis, SAR1 fibroblasts rendered quiescent by culture in the absence of serum were treated with 100 nM 4-HT to activate M+Akt:ER*. Induction of DNA synthesis was assessed by the incorporation of BrdUrd into DNA, which was detected by antibody staining and flow cytometry. Representative data from one of eight separate experiments are shown (Fig. 4, A and B)Citation . In the absence of M+Akt:ER* activation, 16% of SAR1 cells had incorporated BrdUrd (Fig. 4A)Citation . In contrast, after the activation of M+Akt:ER* for 18 h, ~44% of the cells were BrdUrd positive (Fig. 4B)Citation . These data indicate that activation of M+Akt:ER* is sufficient to induce DNA synthesis in Rat1 cells, even in the absence of serum.



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  		Fig. 4. Akt activation is sufficient to induce cell cycle progression in Rat1 cells. SAR1 cells were grown to confluence and rendered quiescent by culture in the absence of serum for 5 days. The cells were then treated with ethanol (A) or with 4-HT (B) for an additional 18 h. The induction of DNA synthesis was assessed by BrdUrd incorporation after activation of M+Akt:ER*. Region 3 (R3) is representative of cells that have incorporated BrdUrd as they progressed through S phase. Cell cycle analysis after activation of M+Akt:ER* is shown. Confluent monolayers of SAR1 cells were rendered quiescent (as above) and then treated with ethanol (C) or with 4-HT (D) for 18 h. The cells were stained with PI, and cell cycle analysis was performed. M1, apoptotic cells; M2, G0-G1 phase cells; M3, S-phase cells; M4, cells in the G2-M phase of the cell cycle.

 
To confirm the data obtained by BrdUrd labeling, SAR1 cells as well as the pooled population of Rat1::M+Akt:ER* cells were rendered quiescent and M+Akt:ER* activated. The cells were stained with PI, and cell cycle analysis was performed using flow cytometry. Representative data of five separate experiments for the SAR1 population are shown (Fig. 4, C and D)Citation . Prior to activation of M+Akt:ER* (Fig. 4C)Citation , ~83% of the cells were in G0-G1 phase (M2), 11% of the cells were in S phase (M3), and 5% of the cells were in G2-M phase (M4). In marked contrast, after activation of M+Akt:ER* (Fig. 4D)Citation , ~48% of the cells were in G0-G1 (M2), 30% of the cells were in S phase (M3), and 19% of the cells were in G2-M phase (M4). Similar results were obtained from the pooled population (data not shown). These data confirm the results of the BrdUrd experiment that activation of M+Akt:ER* is sufficient to induce the progression of serum-deprived Rat1 cells through the cell cycle.

M+Akt:ER* Activation Induces Cyclin E/cdk2 Kinase Activity and Degrades p27Kip1.
The cell cycle is regulated through the periodic synthesis and destruction of cyclins that associate with and regulate the activity of cdks. The activity of the cyclin E/cdk2 complex is required for the G1 to S-phase transition (51 , 52) . The activity of this complex is regulated both by the levels of expression of cyclin E protein as well as by the Cip/Kip family of cdk inhibitors, which bind directly to and inhibit cyclin E/cdk2 (53) . Expression of p27Kip1 is growth inhibitory, and genetic evidence suggests that mice lacking p27Kip1 are abnormally large, have multiple organ hyperplasia, and are predisposed to pituitary tumors (54, 55, 56) . Therefore, to address the molecular mechanism by which activation of M+Akt:ER* promoted the entry of cells into S phase, SAR1 cells as well as the pooled population of Rat1::M+Akt:ER* cells were used to determine the levels of key regulators of the G1-S-phase transition. Both the pooled population and SAR1 cells gave similar results. Representative data for cyclin E expression, the kinase activity of the cyclin E/cdk2 complex, as well as the level of p27Kip1 expression in SAR1 cells after activation of M+Akt:ER* are shown (Fig. 5)Citation . Consistent with the entry of cells into DNA synthesis in Fig. 4Citation , cyclin E/cdk2 kinase activity was found to be increased after activation of M+Akt:ER*. However, the levels of cyclin E in complex with cdk2 were unchanged, indicating that increased cyclin E expression was not responsible for the increased cdk2 kinase activity. In contrast, the level of p27Kip1 was found to be significantly decreased after the activation of M+Akt:ER*. Our findings are consistent with previous work that suggests a role for the PTEN/PI3'-kinase pathway in regulating p27Kip1 expression (57) . Furthermore, our results directly suggest that the effects of the PTEN/PI3'-kinase pathway on p27Kip1 may be mediated through Akt. Because Rat1 cells do not express p21Cip1 (58) , the loss of p27Kip1 from the cyclin E/cdk2 complex likely plays an important role in the M+Akt:ER*-induced entry of cells into S phase. Consequently, the repression of p27Kip1 expression may be a contributing factor to the oncogenic transformation of cells by Akt.



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  		Fig. 5. Activation of M+Akt:ER* induces cdk2 kinase activity and loss of p27Kip1 from the cdk2 complex. Quiescent SAR1 cells were treated with 4-HT to activate M+Akt:ER* for the number of hours indicated, at which point the cells were lysed and cdk2 kinase activity was determined using an immune-complex kinase assay with histone H1 (H1) as a substrate. As a control, cells were treated with ethanol and cultured for 24 h (24-) . The levels of cyclin E, cdk2 in the immunoprecipitates, and the expression of p27Kip1 in cell lysates were determined by Western blot.

 
M+Akt:ER* Activates the ERK/MAP Kinase Pathway.
Because the Raf-MEK-ERK/MAP kinase pathway has been shown to be oncogenic in Rat1 cells, we wanted to determine whether the p42/p44 ERK/MAP kinases were activated in response to M+Akt:ER* activation. Confluent cultures of quiescent SAR1 fibroblasts were treated with 100 nM 4-HT to activate M+Akt:ER* for various lengths of time from 5 min to 3 h, and the activity of ERK1 and ERK2 was determined by Western blot using a phospho-specific anti-active ERK antibody. In response to M+Akt:ER* activation, ERK1 and ERK2 were activated between 30 min and 1 h, with maximum activation in this experiment occurring after ~3 h (Fig. 6A)Citation . In parental Rat1 cells, 4-HT alone did not induce the ERK/MAP kinase pathway (data not shown). The rapid time course of ERK/MAP kinase activation after activation of M+Akt:ER* suggests a direct stimulation of this pathway but does not rule out other possibilities, such as the rapid release of autocrine growth factors.



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  		Fig. 6. M+Akt:ER* activates ERK/MAP kinase in a MEKdependent manner. A, confluent monolayers of SAR1 cells were cultured in the absence of serum for 4 h and were subsequently treated with 4-HT for various times from 5 min to 3 h as indicated. ERK/MAP kinase activity was determined by Western blot analysis with a monoclonal anti-active-phospho-ERK/MAP kinase antibody. B, to determine whether activation of ERK/MAP kinase by M+Akt:ER* was dependent on MEK activity, an immune-complex kinase assay for ERK/MAP kinase activity was performed. SAR1 cells were cultured as above and treated with 4-HT (Akt) or PDGF (10 ng/ml) in the absence (DMSO as solvent control) or the presence of the MEK inhibitor PD098059. The p44 isoform of ERK/MAP kinase was immunoprecipitated with a polyclonal anti-p44 ERK/MAP kinase antibody. The kinase assay was subsequently Western blotted with a polyclonal anti-p44 ERK/MAP kinase antibody to determine the amount of p44 ERK/MAP kinase in each reaction. C, confluent (Con.) and subconfluent (Sub.) monolayers of parental Rat1 cells and Rat1 cells expressing constitutively active Akt (myrAkt) were harvested, and the activity of the p42/p44 ERK/MAP kinases was measured using an immune-complex kinase assay using myelin basic protein as a substrate. As a control, SAR1 cells that were either untreated or treated with 4-HT (100 nM) were harvested. The kinase reactions were subsequently reprobed with antisera that recognize p42/p44 ERK/MAP kinase to determine equal loading of the reactions. IP, immunoprecipitation; WB, Western blot.

 
To determine whether the M+Akt:ER*-induced activation of the ERK/MAP kinase pathway was dependent on MEK, confluent monolayers of quiescent SAR1 fibroblasts were treated with a pharmacological inhibitor of MEK PD098059 for 15 min. The cells were then stimulated with PDGF or with 4-HT. PDGF is known to activate the ERK/MAP kinases in a MEK-dependent manner and therefore serves as an appropriate positive control. Both PDGF and activated M+Akt:ER* were able to rapidly activate the ERK/MAP kinases in these cells (Fig. 6B)Citation . In both cases, the activation of the ERKs was inhibited by PD098059. Therefore, we conclude that the activation of ERK/MAP kinase activity by M+Akt:ER* is dependent upon the activity of MEK.

To determine whether the observed activation of ERK/MAP kinase was a direct result of M+Akt:ER* activation or an unusual property of the Akt:ER* fusion protein, Rat1 cells expressing a constitutively active form of Akt (myrAkt) were generated. Confluent as well as subconfluent cultures of these cells expressing myrAkt and SAR1 cells cultured with 4-HT were found to have elevated levels of ERK/MAP kinase activity (Fig. 6C)Citation . Consequently, these data indicate that activation of p42/p44 ERK/MAP kinases in Rat1 cells is a property of constitutive and conditionally active forms of Akt.

Morphological Transformation by M+Akt:ER* Is Not MEK Dependent.
To determine whether the effects of Akt on cell morphology were mediated through the ERK/MAP kinase pathway, we generated SAR1 cells that also express EGFP{Delta}Raf-1:AR, a conditionally active form of Raf that is regulated by androgens and their analogues. In these cells, we have the capacity to activate M+Akt:ER using 4-HT, EGFP{Delta}Raf-1:AR using testosterone, and both proteins by the co-addition of 4-HT and testosterone. Activation of either Raf or Akt elicited morphological alterations in these cells (Fig. 7, A–C)Citation . Pre-addition of PD098059 to inhibit MEK significantly inhibited the effects of Raf activation on Rat1 cell morphology (Fig. 7Citation , compare B and E). However, in this experiment, PD098059 had little or no effect on the ability of Akt to elicit morphological alterations in these cells (fig. 7Citation , compare C and F). Examination of data from nine separate experiments indicated that occasionally a modest effect of PD098059 on Akt-induced morphological effects was observed but always much less than the effects of the compound on Raf-transformed cells. These data suggest that the ability of Akt to activate the ERK/MAP kinase pathway is likely dispensable for morphological transformation. As yet we have not addressed whether the ERK/MAP kinase pathway participates in the effects of Akt on apoptosis or cell cycle progression.



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  		Fig. 7. Morphological transformation of cells by M+Akt:ER* does not require MEK activity. Confluent cultures of SAR1 fibroblasts infected with eGFP{Delta}Raf: AR were split 1:3 and treated with DMSO or PD098059 (PD) for 15 min. Subsequently, 300 nM testosterone to activate EGFP{Delta}Raf:AR or 100 nM 4-HT to activate M+Akt:ER*, or ethanol as solvent control, were added to cells, and the cells were cultured for an additional 18 h.

 

   Discussion
TOP
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 References
 
Considerable evidence indicates that PI3'-kinase regulated signaling pathways play an important role in a variety of cellular processes (59, 60, 61) and that the protein kinase Akt is an important mediator of these effects (21, 22, 23, 24, 25, 26) . For example, induction of cyclin D1 by growth factors and oncogenes in NIH 3T3 cells is PI3'-kinase dependent, thus suggesting that the PI3'-kinase/Akt pathway may play an important role in the control of cell cycle progression (62 , 63) . Moreover the role of PI3'-kinase in the suppression of apoptosis and the ability of Akt to directly phosphorylate key mediators of the apoptotic response provide strong circumstantial evidence for the importance of the PI3'-kinase-Akt pathway in the aberrant behavior of cancer cells.

Direct evidence for an important role of PI3-kinase and Akt in oncogenic transformation may be inferred from the observation that activated forms of these proteins have been isolated from acutely transforming retroviruses of mice and chickens. An avian retrovirus encoding p3K, an activated form of the catalytic subunit of PI3'-kinase, causes hemangiosarcomas in chickens and transforms chick embryo fibroblasts in culture (64) . A murine retrovirus encoding v-Akt causes T-cell lymphomas (7) and accelerates the development of leukemias in severe combined immunodeficient mice (65) , and overexpression of Akt2 in murine fibroblasts elicits cellular transformation (66) . Furthermore, a mutant form of the p85 regulatory subunit of PI3'-kinase (p65-PI3'-kinase; Ref. 67 ), as well as a conditionally active form of p110 (p110:ER; Ref. 68 ), promotes cellular transformation in mammalian cells in culture.

A role for the PI3'-lipids in human cancer is most strongly implied by the fact that PTEN, a lipid phosphatase that selectively dephosphorylates the 3'-position of the inositol headgroup, is a tumor suppressor gene that is deleted or mutated in both sporadic cancer and in inherited cancer predisposition syndromes. For example, glioblastomas lacking PTEN display elevated PI3'-lipid production and a correspondingly high level of Akt activity (69) . In addition, a number of ovarian cancers display amplification of the gene encoding the p110-catalytic subunit of PI3'-kinase (70) . Various Akt isoforms have been reported to be amplified or overexpressed in breast cancer cells (9) , gastric adenomas (5) , ovarian (41) , and pancreatic (42) cancers. Finally the reported ability of oncogenic Ras to activate PI3-kinase in vitro (71) may provide another link between this signaling pathway and alterations detected in human cancer.

To further study the effects of Akt in mammalian cells, we have established a conditional transformation system for this oncogene using M+Akt:ER, which requires the estrogen analogue 4-HT for its activity. Our results suggest that Akt may activate a number of pathways leading to oncogenic transformation of Rat1 cells. Activation of Akt in Rat1 cells elicited significant alterations in cell morphology, loss of contact inhibition, promoted anchorage-independent proliferation in agarose, protected cells from apoptosis, and promoted the reentry of serum-deprived cells into the cell division cycle.

Progression of cells into S phase requires the activity of cyclin E/cdk2 complexes. The ability of Akt to promote cell cycle progression correlated with its ability to repress the expression of p27Kip1, an inhibitor of the cyclin E/cdk2 complex (72 , 73) . Proliferating cells (74) as well as many types of human cancers have reduced expression of p27Kip1 (75, 76, 77, 78) . In addition, there is mounting evidence to suggest that the progression and prognosis of many forms of cancer are inversely correlated to the levels of p27Kip1 (79, 80, 81, 82) . Expression of p27Kip1 in MCF7 cells reduced their capacity for anchorage-independent cell growth and their tumorigenic capacity in xenografts (83) . However, the mechanisms that influence p27Kip1 expression in tumors have not been extensively explored. Indirect evidence suggests the involvement of the PTEN-PI3'-kinase-Akt pathway in certain circumstances. Ectopic expression of PTEN in glioblastoma cells that lack PTEN expression led to decreased Akt activity and increased p27Kip1 expression and localization in cyclin E/cdk2 complexes (84) . Furthermore Pten-/- embryonic stem cells have increased PI3'-lipid production, a high level of Akt activity, and decreased p27Kip1 expression (85) . However, it has not been demonstrated previously that Akt can directly influence p27Kip1 expression. At present, it is not clear whether decreased p27Kip1 expression is a consequence of a direct effect of Akt on p27Kip1 or whether it may be a consequence of induced cyclin A synthesis. The ubiquitination and subsequent destruction of p27Kip1 is reported to be initiated by cdk2-mediated phosphorylation of p27Kip1 on Thr-187 (74 , 86) . If Akt activation leads to elevated cyclin A expression, it might provoke sufficient cdk2 activation to promote p27Kip1 destruction. Alternatively, Akt may activate a signaling pathway that directly represses p27Kip1 expression. Analysis of the effects of Akt on p27Kip1 mRNA and protein expression as well as the use of various p27Kip1 mutants will allow this question to be addressed.

Perhaps surprisingly, we observed that both constitutive and conditionally active forms of Akt activated the ERK/MAP kinase pathway in Rat1 cells. Although the mechanism of activation remains unclear, there is clear precedent from other studies for a role for PI3'-kinase in ERK/MAP kinase activation (87, 88, 89, 90, 91, 92, 93) . For example, PDGF-induced ERK/MAP kinase activation is inhibited by the PI3'-kinase inhibitors wortmannin and LY294002 under conditions where PDGF is limiting (88) . Furthermore, activation of the Raf-MEK-ERK pathway after integrin engagement is also inhibited by PI3'-kinase inhibitors as well as by a dominant-negative form of p85 (94) . In this case, the PI3'-kinase sensitive step is in the activation of Raf-1. Recent evidence suggests Akt may directly phosphorylate Raf-1, leading to its inactivation during differentiation (95 , 96) , which stands in contrast to the observations presented here. There is compelling evidence to suggest that both the PI3'-kinase/Akt pathway as well as the Raf-MEK-ERK pathway are both required for the expression of key cell cycle regulators (63 , 97) . Our data are consistent with these data and further suggest that the cell type-specific interplay or cross-talk between these oncogenic pathways may play an important role in cell proliferation, differentiation, and transformation. Our future studies will address whether Akt directly phosphorylates Raf-1 in Rat1 cells and determine the effects of Akt on other components of the Ras-activated Raf-MEK-ERK pathway. Despite the ability of Akt to activate the ERK/MAP kinase pathway, it is clear that other Akt-activated pathways must contribute to the effects observed in Rat1 cells because the MEK inhibitor PD098059 had only a modest effect on Akt-induced alterations in cell morphology. To address this, we are currently examining the phosphorylation of other putative Akt targets in Rat1 cells.

The utility of steroid hormone-regulated protein kinases to study intracellular signaling pathways is now well established. In addition, the ability to use steroid hormone binding domains for androgens, mineralocorticoids, progesterone, and others allows cell lines to be constructed in which different signaling pathways are under the control of different steroid hormones. To this end, we have constructed cells in which Raf or Akt may be activated selectively using testosterone or 4-HT, respectively, or both pathways may be coordinately activated by the addition of both hormones. Such cells will permit the analysis of the effects of signal pathway activation either alone or in conjunction with other signaling pathways. By this means, it will be possible to investigate gene regulation and downstream biological effects that occur because of cooperative activation of multiple signaling pathways.


   Materials and Methods
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 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
References
 
Construction of Retrovirus Expression Vectors.
The construction of retrovirus vectors for the expression of M+Akt:ER* in mammalian cells has been described previously (44) . To construct an androgen-regulated form of Raf-1, DNA sequences encoding the hbAR, supplied by Dr. Hartmut Land (University of Rochester School of Medicine and Dentistry, Rochester, NY; Ref. 98 ), were subjected to 10 cycles of PCR to introduce an EcoRI site at the 5' end and a ClaI site at the 3' end of the coding sequence. The resulting PCR fragment was subcloned into pCRII (Invitrogen). Because of the presence of an internal EcoRI site within the sequences encoding hbAR, this plasmid was subjected first to partial digestion with EcoRI, followed by digestion to completion with ClaI to generate a 0.85-kb fragment encoding the entire hbAR region. Retrovirus vectors (pBabepuro3 and pWZLblast3) encoding EGFP{Delta}Raf-1:ER (46 , 99) were digested with EcoRI and ClaI to remove sequences encoding the hormone binding domain of the estrogen receptor. Ligation of the EcoRI-ClaI fragment of hbAR led to the generation of retrovirus vectors encoding EGFP{Delta}Raf-1:AR. When EGFP{Delta}Raf-1:AR was expressed in cells, the activity of the MEK/MAP kinase pathway is regulated by the addition or subtraction of testosterone or the synthetic androgen analogue R1881 (DuPont NEN) to the cell culture media (98) . Further details of these constructions are available on request.

Cell Culture, Retrovirus Production, and Infection.
All cells were cultured in phenol red-free DMEM supplemented with 10% fetal bovine serum, penicillin, and streptomycin at 37°C in a humidified atmosphere containing 5% (v/v) CO2. Cells were photographed with a Nikon TMS photomicroscope as described previously (45) . Testosterone (Sigma) and 4-HT (Research Biochemicals) were prepared as 1 mM stocks in ethanol, stored at -20oC, and used at the indicated concentrations. Retrovirus stocks were obtained by Lipofectamine (Life Technologies, Inc.)-mediated transfection of the appropriate vectors into Bosc23 cells, as described previously (100) . Target cells were infected and then cultured in medium containing 2–10 µg/ml puromycin or 12–25 µg/ml blasticidin (Sigma), depending on the vector, to select for virus-infected cells. Standard virus stocks gave rise to >=106 puromycin- or blasticidin-resistant colonies/ml of virus. After drug selection, cells were pooled, expanded, and tested for the expression M+Akt:ER* or EGFP{Delta}Raf-1:AR proteins by Western blotting and, where appropriate, by FACScan at 490 nm. Under these conditions, >=95% of the pooled blasticidin-resistant cells expressed the EGFP{Delta}Raf-1:AR fusion proteins. Rat1::M+Akt:ER* fibroblasts are a pooled population of Rat1 fibroblasts expressing M+Akt:ER*.

Soft agar-selected Rat1::M+Akt:ER* colonies were used to derive the SAR1 clonal cell line. To generate these lines, cells from the pooled population were plated and cultured in DMEM containing 0.35% (w/v) agarose in the presence of 4-HT until colonies were visible. Individual colonies were selected at random, the agarose was removed by pipetting with media, and the colony was plated onto the well of a 24-well dish for subsequent expansion.

SAR1 cells were superinfected with a virus encoding EGFP{Delta}Raf-1:AR to produce the SAR1-Raf cells, which express the 4-HT-responsive M+Akt:ER* and androgen-responsive EGFP{Delta}Raf-1:AR. These cells were cloned as single cells using a fluorescence-activated cell sorter (Becton Dickinson).

Confluent monolayers of cells were rendered quiescent by culture in DMEM containing penicillin, streptomycin, 25 mM HEPES, and 2.5/500 mg/l linoleic acid/BSA complex, respectively for the times indicated.

Preparation of Cell Extracts and Analysis by Western Blot.
Triton X-100 soluble cell lysates were prepared as described (45) . Cells were lysed in Gold lysis buffer [1% (v/v) Triton X-100, 20 mM Tris (pH 8.0), 137 mM NaCl, 15% glycerol, and 5 mM EDTA] plus protease inhibitors (1 µM phenylmethylsulfonyl fluoride and 10 µM pepstatin) and phosphatase inhibitors (1 mM EGTA, 10 mM NaF, 1 mM tetrasodium PPi, 100 µM ß-glycerophosphate, and 1 mM sodium orthovanadate), and protein concentrations were measured using the BCA protein assay kit (Pierce). Aliquots of cell lysates were electrophoresed through polyacrylamide gels and Western blotted onto Immobilon P polyvinylidene difluoride membranes (Millipore). Western blots were probed with the appropriate dilutions of primary antibodies for at least 1 h at room temperature. Anti-ER ({alpha}ER) and anti-p44 ERK/MAP kinase were from Santa Cruz Biotechnology; anti-Akt (PKB{alpha}) was from Upstate Biotechnology; and the anti-phospho-Ser-473-specific antibody was from New England Biolabs. Antigen-antibody complexes were detected using the appropriate secondary antibody or protein A coupled to horseradish peroxidase and visualized using the enhanced chemiluminescence detection system (ECL; Amersham).

DNA Synthesis and Apoptosis Assays.
DNA synthesis was assessed by the incorporation of BrdUrd into cellular DNA as described previously (99 , 101) . Briefly, cells were rendered quiescent by culture in DMEM + BSA/linoleic acid for 5 days with media changed every 24 h. After quiescence, the cells were treated as described in the text. BrdUrd was added to the cells to a final concentration of 50 µM, and the incubation continued for an additional 18 h. Ethanol-fixed cells were stained with an anti-BrdUrd antibody (Becton Dickinson). After staining, the cells were washed and analyzed using a Becton Dickinson FACScan.

Apoptosis was assessed after activation of M+Akt:ER* by staining with Annexin V-coupled to FITC, as detailed in the ApoAlert Apoptosis kit protocol (Clontech Laboratories). To determine whether cells were undergoing apoptosis, flow cytometry was used to determine Annexin V and PI staining. Live cells are found in the lower left quadrant, cells in the early stages of apoptosis are found in the lower right quadrant, and cells late in the apoptotic process are found in the upper right quadrant (102 , 103) . In addition, PI staining for cell cycle analysis and scoring of the sub-G0-G1 population was also used.

Immune-Complex Kinase Assays.
Akt immune-complex kinase assays were performed as described previously (31) . Briefly, cells were lysed in Gold lysis buffer, plus protease and phosphatase inhibitors, and Akt was immunoprecipitated with a polyclonal anti-Akt (PKB{alpha}) antibody (UBI). Immunoprecipitates were washed twice with lysis buffer, twice in buffer containing 25 mM HEPES (pH 7.2), 1 M NaCl, 0.1% (w/v) BSA, 10% (v/v) glycerol, and 1% (v/v) Triton X-100, and twice again with kinase reaction buffer containing 20 mM HEPES (pH 7.2), 10 mM MgCl2, 10 mM MnCl2, 1 mM DTT, 5 µM ATP, and 0.2 mM EGTA. Akt kinase reactions were performed in 20-µl reaction buffer supplemented with 2 µg of protein kinase A inhibitor protein (PKI; Sigma), 10 µCi [{gamma}-32P]ATP (Amersham), and 500 ng of histone H2B (Boehringer Mannheim). The reactions were performed for 30 min at 30oC and quenched with 5x sample buffer containing ß-mercaptoethanol and boiled for 5 min. Kinase reactions were electrophoresed and Western blotted, and the phosphorylation of substrates was quantitated using a Molecular Dynamics Storm phosphorimager. After quantitation, the Western blots were probed with the appropriate antibody to detect the amount of protein in each lane.

ERK/MAP kinase immune-complex kinase assays were performed as described previously (45) . In brief, after cell lysis, p44 ERK/MAP kinase was immunoprecipitated with a polyclonal anti-p44 antibody (C16; Santa Cruz Biotechnology). The immunoprecipitates were washed three times with lysis buffer, once in lysis buffer plus 0.5 M NaCl, and once in ERK/MAP kinase wash buffer containing 25 mM Tris, 40 mM MgCl2, 137 mM NaCl, and 10% (v/v) glycerol. The reaction was carried out in 20 µl of kinase reaction buffer containing 1 M HEPES (pH 7.4), 1 M MgCl2, 1 mM ATP, 2 mg/ml myelin basic protein (Sigma), and 10 µCi [{gamma}-32P] ATP (Amersham). The reactions were performed for 30 min at 30oC and quenched with 5x sample buffer containing ß-mercaptoethanol and boiling for 5 min, analyzed as described above.

For cdk2 immune-complex kinase assays, cells were lysed in NP40 lysis buffer [50 mM HEPES (pH 7.5), 0.1% (v/v) NP40, and 250 mM NaCl]. The lysates were precleared with protein A-Sepharose (Sigma) and immunoprecipitated with a rabbit polyclonal anti-cdk2 antibody (UBI). Immunoprecipitates were washed three times in NP40 lysis buffer and once in cdk2 kinase reaction buffer [50 mM Tris (pH 7.4), 10 mM MgCl2, and 1 mM DTT]. The reaction was carried out at 30oC for 30 min in 40 µl of kinase reaction buffer containing 2.5 µg of histone H1, 10 µCi [{gamma}-32P]ATP, and 0.01 mM ATP. The reactions were quenched in 5x sample buffer containing ß-mercaptoethanol and boiling for 5 min, analyzed as above.


   Acknowledgments
 
We thank Drs. Douglas Woods, Emma Lees, David Parry, David Stokoe, and Harmut Land, as well as the members of the McMahon lab, for the provision of reagents and for useful comments and discussions over the course of this work.


   Footnotes
 
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 Supported by the DNAX Research Institute and the University of California, San Francisco Cancer Research Institute (M.M.), NIH Grant DK34926 (to R.R.), and NIH Postdoctoral Training Grant CA092770-23 (to A.M.M.). Back

2 To whom requests for reprints should be addressed, at Cancer Research Institute, UCSF/Mt. Zion Cancer Center, 2340 Sutter Street, San Francisco, CA 94115. Phone: (415) 502-5829 or (415) 502-1317; Fax: (415) 502-3179; E-mail: mcmahon{at}cc.ucsf.edu Back

3 The abbreviations used are: PKA, protein kinase A; PKC, protein kinase C; PKB, protein kinase B; PH, pleckstrin homology; PI3'-kinase, phosphatidylinositol 3'-kinase; ER, estrogen receptor; AR, androgen receptor; 4-HT, 4-hydroxytamoxifen; cdk, cyclin-dependent kinase; ERK, extracellular signal-regulated kinase; MAP, mitogen-activated protein; MEK, MAP/ERK kinase; PI, propidium iodide; SAR1, soft agar-derived Rat1::M+Akt:ER*; BrdUrd, 5-bromodeoxyuridine; PDGF, platelet-derived growth factor; hbAR, hormone binding domain of the human AR. Back

Received for publication 2/ 4/00. Revision received 4/27/00. Accepted for publication 5/ 1/00.


   References
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 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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