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Molecular Cancer Research 3:297-305 (2005)
© 2005 American Association for Cancer Research

Signaling and Regulation

Requirement of Activated Cdc42-Associated Kinase for Survival of v-Ras-Transformed Mammalian Cells

Alam Nur-E-Kamal1, Ailing Zhang1, Susan M. Keenan1, Xin I. Wang1, Jabed Seraj2, Takaya Satoh3, Sally Meiners1 and William J. Welsh1

1 Department of Pharmacology, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey; 2 Cancer Therapeutics Research Team, Johnson and Johnson Pharmaceutical Research and Development, Raritan, New Jersey; and 3 Division of Molecular Biology, Department of Molecular and Cellular Biology, Kobe University Graduate School of Medicine, Chuo-Ku, Kobe, Japan

Requests for reprints: Alam Nur-E-Kamal, Department of Pharmacology, University of Medicine and Dentistry of New Jersey, 675 Hoes Lane, Piscataway, NJ 08854. Phone: 732-235-0106; Fax: 732-235-4073. E-mail: nurekasa{at}umdnj.edu


   Abstract
Top
 Notes
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
Activated Cdc42-associated kinase (ACK) has been shown to be an important effector molecule for the small GTPase Cdc42. We have shown previously an essential role for Cdc42 in the transduction of Ras signals for the transformation of mammalian cells. In this report, we show that the ACK-1 isoform of ACK plays a critical role in transducing Ras-Cdc42 signals in the NIH 3T3 cells. Overexpression of a dominant-negative (K214R) mutant of ACK-1 inhibits Ras-induced up-regulation of c-fos and inhibits the growth of v-Ras-transformed NIH 3T3 cells. Using small interfering RNA, we knocked down the expression of ACK-1 in both v-Ha-Ras-transformed and parental NIH 3T3 cells and found that down-regulation of ACK-1 inhibited cell growth by inducing apoptosis only in v-Ha-Ras-transformed but not parental NIH 3T3 cells. In addition, we studied the effect of several tyrosine kinase inhibitors and found that PD158780 inhibits the kinase activity of ACK-1 in vitro. We also found that PD158780 inhibits the growth of v-Ha-Ras-transformed NIH 3T3 cells. Taken together, our results suggest that ACK-1 kinase plays an important role in the survival of v-Ha-Ras-transformed cells, suggesting that ACK-1 is a novel target for therapies directed at Ras-induced cancer.


   Introduction
Top
 Notes
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
Ras is a multieffector signaling molecule that has been implicated in the regulation of many cellular functions, including cell growth, differentiation, apoptosis, movement, and transformation (1, 2). Mutations in Ras genes that encode constitutively active proteins have been reported in at least 30% of human cancers (3, 4); indeed, overexpression of Ras has been reported in various types of breast cancer and leukemia (5, 6). Furthermore, functional activation of a nononcogenic form of Ras contributes to the molecular pathogenesis of brain tumors and breast cancers (7-9). Taken together, these observations suggest that diverse agonists and receptors are able to use the activation of Ras as a common mechanism for the progression of cancer and indicate that contribution of Ras activation in molecular pathogenesis is more complex than initially anticipated. Although Ras has recently been shown to activate multiple signaling pathways through specific effectors, the molecular mechanism of the transduction of Ras-induced transformation signals remains unclear.

Several lines of evidence support the hypothesis that the Rho-GTPase family member Cdc42 is essential for Ras-induced transformation of mammalian cells. For example, blocking Cdc42 signaling by overexpressing a dominant-negative N17Cdc42 mutant of Cdc42 (10) or by using a Cdc42-specific polypeptide inhibitor [derived from activated Cdc42-associated kinase (ACK-1); ref. 11] reverses Ras-induced cellular transformation. Cells stably expressing a constitutively activate mutant (F28L) of Cdc42 exhibit several hallmarks of transformation-induced contact inhibition, including lower dependence on serum for growth and anchorage-independent growth (12). Further evidence for a role of Cdc42 as well as ACK in tumor invasion is provided by the fact that spreading of melanoma cells due to activation of melanoma chondroitin sulfate proteoglycan is dependent on active Cdc42 and ACK and the subsequent tyrosine phosphorylation of the docking protein p130Cas (13). A role for Cdc42 in the control of normal cell growth is also supported by the overexpression of constitutively active mutants (G12V or Q61L) of Cdc42 in NIH 3T3 cells, which stimulated cell growth, caused loss of serum dependence, and showed increased saturation density (14). Collectively, this evidence points to a signaling pathway from the cell surface, through Ras, for Cdc42 and ACK in controlling cell growth and tumor formation.

Two isoforms of ACK, ACK-1 and ACK-2, have been identified. ACK-1 is a 120-kDa protein that was originally cloned from a human brain cDNA library and was characterized as a potential effector for Cdc42 (15). More recently, a second ACK, ACK-2 with a molecular mass of 85 kDa, was cloned from a bovine brain cDNA library (16). Overexpression of ACK-2 results in activation of c-Jun NH2-terminal kinase indicative of a potential role for ACK-2 in the cellular stress response (17). Recently, ACK-1 and ACK-2 were found to coimmunoprecipitate with clathrin and to be localized in the nucleus of fibroblasts (18, 19). However, only one gene and one protein product for ACK have thus far been identified in human cells, suggesting that ACK-1 and ACK-2 may represent species-specific variants.

We have identified a nuclear export signal in ACK and have found a Cdc42-dependent nuclear localization of ACK-1 in glial cells (20). The high-resolution crystal structure of the Cdc42-binding domain (highly conserved in both ACKs) complexed with ACK-1 reveals key amino acids involved in the ACK-Cdc42 interaction (21). ACK-1 was recently shown to interact with the SH3 domain of Hck, a member of the Src kinase family (22). Moreover, epidermal growth factor activates the ACK-1/Dbl pathway, where Dbl is involved in the regulation of cell migration (23-25), and the Drosophila orthologue of ACK-1 is involved in axonal guidance (26). These observations indicate an important role of ACK in transducing Ras-Cdc42 signals for various cellular functions.

In the current study, we investigated the role of ACK in the transduction of Ras-Cdc42 signals in mammalian cell transformation. We now show that ACK-1 is required for v-Ha-Ras-induced up-regulation of c-fos. Furthermore, down-regulation of ACK-1 by small interfering RNA (siRNA) induced apoptosis in v-Ras-transformed NIH 3T3 cells but not in parental NIH 3T3 cells. Using an ACK kinase domain expressed in bacteria, we identified tyrosine kinase inhibitors that inhibited ACK-1 kinase activity and also inhibited the growth of v-Ras-transformed cells. Collectively, these results indicate that Ras transformants, but not normal cells, are highly reliant on the ACK-1 kinase for both survival and growth. These data provide the first evidence demonstrating an important role of ACK in transducing Ras signals for transformation of mammalian cells.


   Results
Top
 Notes
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 Acknowledgements
 References
 
ACK-1 Is Required for Transducing v-Ras Signals in Mammalian Cells
We have shown previously that activation of Ras increases the level of GTP-bound Cdc42 in NIH 3T3 and PC12 cells (11). Therefore, we evaluated whether activation of Ras induces phosphorylation of ACK-1, which acts downstream of activated Cdc42 (15). We now report that expression of v-Ha-Ras in NIH 3T3 cells induces phosphorylation of ACK-1 (Fig. 1A), whereas coexpression of a dominant-negative mutant of Cdc42 blocked v-Ha-Ras-induced phosphorylation of ACK (Fig. 1A). This suggests that the Ras signal for ACK-1 phosphorylation is transduced through Cdc42. Activation of either Ras or Cdc42 has been shown to induce the expression of early-response genes, such as c-fos and JNK (27, 28). Although Ras transformation of mammalian cells has been shown to be transduced through Cdc42 (10, 11), it remains unclear whether the Ras signal for the expression of c-fos is also coupled to Cdc42 and/or ACK.



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  		FIGURE 1. ACK-1 transduces Ras signals for transformation of mammalian cells. A. Involvement of Cdc42 in transducing Ras signals in inducing phosphorylation of ACK-1. NIH 3T3 cells were cultured in DMEM containing 10% FCS. Cells were transfected with vector alone, v-Ha-Ras, V12Cdc42, or v-Ha-Ras/N17Cdc42 constructs. Cells were lysed and ACK was immunoprecipitated as described in Materials and Methods. Proteins obtained in the immunoprecipitate were separated by SDS-PAGE (8%), and Western blotting was done using antibodies against phosphotyrosine (P-Tyr). The amount of ACK-1 protein in each sample was determined by blotting the same membrane with antibodies against ACK-1. B. Ras-Cdc42 signals for up-regulation of c-fos are transduced through ACK-1. NIH 3T3 cells were cultured as described above. Cells were transfected with pMV7 (vector) as control, pMV7-ACKKR, pMV7-ACKLF, v-Ras cDNA or were cotransfected with pMV7-ACKKR and v-Ras cDNA using Cellfectin reagent. After 4 hours, cells were collected and lysed in Laemmli SDS sample buffer. Proteins were separated by SDS-PAGE (10%), and Western blotting was done using c-fos or Ha-Ras antibodies. Equal loading of total protein was confirmed by blotting the membrane with actin antibodies. Arrows, positions of c-fos and actin. C. Overexpression of the kinase mutant (K214R) of ACK-1 inhibits growth of v-Ras-transformed cells. Normal and v-Ha-Ras-transformed NIH 3T3 cells were transfected with Cellfectin alone or were complexed with pMV7 (control), pMV7-ACKLF, or pMV7-ACKKR. After 4 hours, the transfection reagent–containing medium was replaced with DMEM containing 10% FCS, and cells were incubated under standard cell culture conditions. After 48 hours, cells were collected by trypsinization and their number was counted using a hemocytometer and compared with the number obtained for a vector alone transfection sample (bottom). In a parallel experiment, cell lysates were separated by SDS-PAGE (10%). Western blotting was done using antibodies against Ha-Ras (top). Equal loading of protein was confirmed by blotting the membrane with anti-actin antibodies.

 
We next evaluated whether ACK-1 is involved in transducing Ras signals for c-fos expression. First, we transfected a v-Ha-Ras (constitutively active) expressing plasmid into NIH 3T3 cells. Expression of v-Ha-Ras up-regulated c-fos, whereas transfection of vector alone had no effect on c-fos levels (Fig. 1B). Cotransfection of a kinase-dead mutant (K214R) of ACK-1 with the v-Ras construct into NIH 3T3 cells inhibited v-Ras-induced up-regulation of c-fos (Fig. 1B). We then expressed the K214R mutant of ACK in v-Ha-Ras-transformed and parental NIH 3T3 cells. K214R significantly inhibited the growth of v-Ha-Ras-transformed cells. Interestingly, the expression of the K214R had no effect on the expression of c-fos, or on the growth of normal NIH 3T3 cells (Fig. 1B and C), despite similar levels of K214R expression in transformed and parental NIH 3T3 cells (data not shown). Although the constitutively active mutant of ACK-1 (L543F) induced c-fos expression in NIH 3T3 cells (Fig. 1B), the L543F mutant, with similar levels of expression in each cell type (data not shown), had no effect on the growth of parental and v-Ha-Ras-transformed NIH 3T3 cells (Fig. 1C). Transfection of K214R and L543F did not alter the level of Ras expression in v-Ha-Ras-transformed cells (Fig. 1C), suggesting that inhibition of cell proliferation was not due to loss of Ras expression. These results indicate that ACK-1 is involved in transducing Ras signals and that ACK-1-dependent signals play a critical role in growth of v-Ha-Ras-transformed mammalian cells.

ACK Is Required for Survival of v-Ha-Ras-Transformed NIH 3T3 Cells
To further investigate whether ACK-1 is required for growth and survival of v-Ha-Ras-transformed cells, we knocked down the expression of ACK-1 using siRNA. Transfection of ACK-1 siRNA reduced the expression of ACK-1 in a dose-dependent manner; 0.8 nmol/L ACK siRNA reduced the level of ACK-1 significantly in v-Ha-Ras-transformed and parental NIH 3T3 cells (Fig. 2A and B). Under these conditions, we studied the role of ACK-1 in the regulation of growth and survival of v-Ha-Ras-transformed cells. Transfection of ACK-1 siRNA similarly inhibited the growth of v-Ha-Ras-transformed cells in a dose-dependent manner (Fig. 2C), whereas transfection of sense strand of siRNA did not affect the growth of v-Ha-Ras-transformed NIH 3T3 cells (Fig. 2C). However, transfection of ACK-1 siRNA did not affect the growth of parental NIH 3T3 cells (Fig. 2D). Therefore, v-Ha-Ras-transformed cells, but not normal cells, may be dependent on ACK-1-mediated growth and fail to produce sufficient survival signals when the ACK-1-dependent Ras signaling pathway is interrupted. These results suggest an important involvement of ACK-1 in controlling the growth and survival of v-Ha-Ras-transformed mammalian cells.



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  		FIGURE 2. Inhibition of v-Ha-Ras-transformed cell growth by ACK siRNA treatment. v-Ha-Ras-transformed NIH 3T3 (A and C) and parental NIH 3T3 (B and D) cells were cultured in DMEM containing 10% FCS. Cells were treated with Cellfectin, Cellfectin complexed with the sense strand of ACK-1 siRNA, or ACK-1 siRNA. A and B. Down-regulation of ACK by siRNA treatment. After 24 hours of transfection, cells were collected and lysed with Laemmli SDS sample buffer. Proteins were separated by SDS-PAGE and the level of ACK-1 protein was determined by Western blotting using antibodies against ACK-1. Equal loading of total proteins was confirmed by blotting the membrane with actin antibodies. Arrows, positions of ACK-1 and actin. C and D. Inhibition of v-Ha-Ras-transformed cell growth by siRNA treatment. After transfection with siRNA at different concentrations (in nmol/L), v-Ha-Ras-transformed and parental NIH 3T3 cells were trypsinized and collected every 24 hours. Cell numbers were counted in triplicate.

 
We also investigated whether ACK-1 alone can transform NIH 3T3 cells. We developed a stable NIH 3T3 cell line that overexpressed either wild-type ACK-1 or a constitutively activated kinase mutant (L543F) of ACK-1. Neither ACK-1 nor the L543F mutant of ACK-1 produced a transformation phenotype in our transformation assay (data not shown). These results indicate that ACK alone is not sufficient to induce transformation of NIH 3T3 cells.

Down-Regulation of ACK-1 Induces Apoptosis in v-Ha-Ras-Transformed Cells
We next explored whether ACK-1 deficiency induces apoptosis in v-Ha-Ras-transformed cells. To investigate this question, ACK siRNA was transfected into v-Ha-Ras-transformed cells to knockdown the expression of ACK-1 and studied apoptosis. We found that transfection of ACK-1 siRNA induced apoptosis as determined by studying apoptosis markers, such as poly(ADP-ribose) polymerase cleavage (Fig. 3A), cleavage of the inhibitor of caspase-activated DNase (Fig. 3B), release of cytochrome c from mitochondria (Fig. 3D), and fragmentation of chromosomal DNA (Fig. 3C). Interestingly, transfection of ACK siRNA did not block v-Ha-Ras-transformed cells at any particular stage of the cell cycle (data not shown), suggesting that ACK deficiency induced cell death in a cell cycle–independent manner. Collectively, these results suggest that Ras signals transduced through ACK-1 are required to protect v-Ha-Ras-transformed cells from apoptosis.



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  		FIGURE 3. Induction of apoptosis by down-regulation of ACK in v-Ras-transformed NIH 3T3 cells. v-Ha-Ras-transformed NIH 3T3 cells were treated with DMEM (control), Cellfectin, Cellfectin complexed with the sense strand of ACK-1 siRNA, or ACK-1 siRNA. Treatment of cells with DNA topoisomerase II inhibitor, etoposide (VP-16), was done to provide a positive control. After 24 hours, cells were collected. Cells were lysed to get the total cellular proteins or fractionated to get cytoplasmic proteins as described previously (58). Proteins (total cellular and cytoplasmic) were separated by SDS-PAGE, and Western blotting was done using antibodies against poly(ADP-ribose) polymerase (PARP; A), inhibitor of caspase-activated DNase (ICAD; B), and cytochrome c (Cyt C; D). Equal loading of total protein was confirmed by blotting the membrane with antibodies against actin. C. Cells were treated with Cellfectin, Cellfectin complexed with the sense strand of siRNA, or siRNA. Cells were collected after 21 hours. An equal number of untreated (control) and VP-16–treated cells were also collected after 21 hours of incubation. The cytoplasmic fraction was isolated, and DNA fragments were extracted and purified by ethanol precipitation. Isolated DNA fragments were characterized by 1.5% agarose gel electrophoresis. The experiment was repeated thrice showing similar results.

 
Inhibition of the Kinase Activity of ACK-1 by Tyrosine Kinase Inhibitors In vitro
We screened several tyrosine kinase inhibitors to examine their potency in inhibiting the kinase activity of ACK in vitro. The polypeptide (ACKD), which corresponds to the kinase and SH3 domains of ACK-1 (amino acids 101-441), was cloned in a bacterial expression vector, produced as a glutathione S-transferase (GST)-fusion protein (GST-ACKD), and affinity purified. GST-ACKD exhibited autokinase activity as well as phosphorylated myelin basic protein (MBP; Fig. 4A and B). Several tyrosine kinase inhibitors, including PD158780, an inhibitor for the epidermal growth factor receptor (EGFR) and mitogen-activated protein kinase (29), and PD157432, an inhibitor for EGFR (30), were studied for their ability to inhibit the kinase activity of GST-ACKD in vitro. PD158780 has the strongest inhibitory activity, whereas quercetin, genistein, wortmannin, and PD157432 exhibited very weak activity (Fig. 4C and D).



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  		FIGURE 4. Inhibition of ACK kinase activity by kinase inhibitors. A fragment of ACK-1 kinase (ACKD) and its K214R kinase mutant (ACKKR) were produced in E. coli and affinity purified as GST-fusion proteins. Kinase activity of the bacterially produced GST-fusion proteins was assayed using MBP as a substrate. Reaction products were characterized by SDS-PAGE followed by autoradiography (A). ACKD phosphorylated MBP in a dose-dependent manner (B). Arrows, expected positions of ACKD, ACKKR, and MBP. C and D. Effect of kinase inhibitor on the kinase activity of ACK-1. Different kinase inhibitors were added to the ACK-1 kinase reaction as described in Materials and Methods. Phosphorylation of MBP was determined by SDS-PAGE and autoradiography. The level of MBP phosphorylation was determined by scanning MBP bands using Kodak Imaging Station 2000R and plotted as arbitrary units. PD158780 inhibited ACK strongly and in a dose-dependent manner (C). The effect of independent kinase inhibitors at a concentration of 200 nmol/L is shown (D). Each of these experiments was repeated thrice showing similar results.

 
Tyrosine Kinase Inhibitors Inhibit v-Ha-Ras-Transformed NIH 3T3 Cell Growth
The effect of the tyrosine kinase inhibitors PD158780 and PD157432 on the phosphorylation of ACK-1 was investigated. v-Ha-Ras-transformed cells were treated with PD158780 or PD157432 at a concentration of 25 µmol/L for 48 hours. Phosphorylation status of ACK-1 was then determined by immunoprecipitation and Western blotting. It was found that PD158780 inhibited ACK-1 autophosphorylation to a much stronger extent than did PD157432 (Fig. 5A). These results suggest that PD158780 inhibits ACK kinase in v-Ras-transformed cells.



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  		FIGURE 5. Inhibition of v-Ha-Ras-transformed cell growth by PD158780. v-Ha-Ras-transformed cells were cultured in DMEM containing 10% FCS. Cells were treated with solvent (DMSO), PD158780 (25 µmol/L), or PD157432 (25 µmol/L) for 48 hours. Cells were incubated under standard cell culture conditions. For ACK-1 immunoprecipitation, cells were lysed and ACK-1 was immunoprecipitated as described in Materials and Methods. Proteins present in the immunoprecipitate were separated by SDS-PAGE (8%), and Western blotting was done using anti-phosphotyrosine antibody (A). Equal loading of ACK-1 was confirmed by blotting the same membrane with antibodies against ACK-1. For growth inhibition studies, cells were trypsinized and counted every 24 hours. The growth of v-Ras-transformed cells in the presence or absence of PD158780 (B) and PD157432 (C) were plotted.

 
We then examined whether incubation with PD158780 or PD157432 affected the growth of v-Ha-Ras-transformed NIH 3T3 cells. After treating v-Ha-Ras-transformed NIH 3T3 cells with PD158780, we studied cell growth and found that the growth was inhibited in a dose-dependent manner (Fig. 5A), whereas PD157432 did not show any inhibitory effect (Fig. 5B). Although we realize that ACK-1 is not the only potential in vivo target for either inhibitor (both compounds can inhibit EGFR and PD158780 is also an extracellular signal regulated kinase inhibitor), the differential abilities of the inhibitors to modulate ACK-1 phosphorylation and activity correlate strongly with their effects on the growth of v-Ha-Ras-transformed cells (Fig. 5).


   Discussion
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 Acknowledgements
 References
 
The family of Ras GTPases plays a critical role in controlling growth of normal and transformed cells (2-4). Ras has also been shown to play an essential role in maintenance of tumor growth in a mouse model (31). For these reasons, the Ras signaling pathway(s) involved in the regulation of transformation has been a major focus in recent years. Like Ras GTPase, Cdc42 GTPase is involved in the regulation of several normal cellular functions, including maintenance of cell polarity (32, 33). Cdc42 is involved in transducing, at least in part, Ras signals for transformation (10, 11, 20). Therefore, the aim of this study was to identify the Cdc42 signaling pathway(s) involved in transducing the Ras-specific transformation signal.

ACK is an important effector molecule for Cdc42 (15). Activation of the EGFR has recently been shown to activate both Cdc42 and ACK-1 (23). Furthermore, ACK-1 has also been shown to transduce p130Cas-induced metastasis signals in melanoma cells (13), suggesting a possible role of ACK-1 in transducing Ras signals in mammalian cells. To study the role of ACK-1 in transducing Ras signals for transformation, we first showed that introducing a constitutively active v-Ras construct into NIH 3T3 cells establishes a transformed NIH 3T3 cell line (34-36). S.c. injection of these v-Ras-transformed cells produced tumors in nude mice,4 demonstrating that they provide a good in vitro model of cancer cells. Consistent with the hypothesis that ACK-1 is involved in a Ras signaling pathway, we showed that overexpression of a dominant-negative mutant (K214R) of ACK-1 blocks Ras signals for up-regulation of c-fos expression (Fig. 1) and inhibits proliferation of the v-Ha-Ras-transformed cells (Fig. 1). This is the first evidence demonstrating an involvement of ACK-1 in transducing Ras signals and further suggests a likely role of ACK-1 in transducing Ras signals for the transformation of mammalian cells.

Whereas knocking down the expression of ACK-1 in v-Ras-transformed cells induced apoptosis, no induction of apoptosis in ACK-1-deficient parental NIH 3T3 cells was detected. Instead, ACK-1-deficient NIH 3T3 cells continued to proliferate. This suggests that v-Ras-transformed cells are dependent on ACK-1 signaling for growth and survival, whereas ACK-1-dependent signals are dispensable in parental NIH 3T3 cells. To this point, several observations have recently been published, demonstrating that transformed cells become dependent on particular signaling pathways for survival (37-39). These signaling pathways are being targeted to develop therapies for various types of cancers. Our results suggest that ACK-1 is likewise a potential target for the development of a therapy for Ras-induced cancer.

PAK1 has also been shown to be required for Ras-induced transformation (40, 41), but PAK1 (or its activated mutant) alone is not sufficient to produce the transformation phenotype (42). Activated Ras GTPase has been shown to stimulate multiple signaling pathways, including activation of Raf-1-, phosphatidylinositol 3-kinase-, and Ral GDS-mediated pathways (43-47). However, it has been shown that activation of more than one of these pathways is required for transformation of mammalian cells (48-51). ACK-1 may be located downstream of one of these signaling pathways. However, upstream targets of ACK-1 have not yet been identified. Downstream targets of ACK-1 that are involved in transformation of mammalian cells have also not been characterized. Overexpression of ACK activates c-Jun NH2-terminal kinase (17), and ACK regulates expression of EGFR in Caenorhabditis elegans (52) and mammalian cells (53), suggesting a possible role for c-Jun NH2-terminal kinase or EGFR in transformation. We are currently investigating the upstream and downstream targets of ACK-1 involved in Ras-induced transformation.

ACK-1 is a member of the non–receptor tyrosine kinase family (54). Mechanisms of signal transduction involving members of this family seem to be more complex than anticipated previously. Several reports have shown that members of the non–receptor tyrosine kinases "cross-talk" to each other. For example, Fyn can phosphorylate ACK, and ACK can phosphorylate Hck and sorting nexin 9 (22, 53, 55). Furthermore, we have recently found that ACK-1 phosphorylates the DH-PH domain of Tiam1, a protein involved in metastasis.4 These observations suggest that ACK-1 may play a complex role in cell growth and differentiation depending on cell type.

We next used an in vitro assay system to pharmacologically evaluate the role of ACK-1 in transduction of signals associated with cellular transformation. We identified several kinase inhibitors that inhibit ACK-1 kinase activity (Fig. 4). Inhibition of ACK-1 kinase activity by PD158780 was the strongest, and PD158780 also inhibits growth of v-Ras-transformed NIH 3T3 cells. PD157432 exhibited weak inhibitory activity on ACK kinase and did not inhibit the growth of v-Ha-Ras-transformed cells. However, no chemical inhibitor specific for this kinase has been developed yet, although we recently found that PD158780, an ErbB1 inhibitor (IC50, ~13 nmol/L; ref. 56), is able to inhibit ACK-1 as well, but with the IC50 ~200 nmol/L in vitro (Fig. 4). Although it is possible that PD158780-dependent v-Ha-Ras cell death is due to inhibition of kinases other than ACK (e.g., EGFR or extracellular signal-regulated kinase), we show through the use of mutants and siRNA that inhibition ACK alone does induce v-Ha-Ras-transformed cell death. Furthermore, there is consistency between the effects of the small molecule compounds on the inhibition of ACK activity and on cell death. Collectively these data suggest that ACK-specific kinase inhibitors could indeed prohibit cellular growth by inducing apoptosis of v-Ha-Ras-transformed cells.

We investigated whether PD158780 does, in fact, fit into the active site of the kinase domain of ACK. The recent publication of the high-resolution X-ray crystal structure of ACK-1 reveals, as expected, the typical protein kinase fold: a bilobate structure consisting of a ß-sheet dominated NH2-terminal lobe and a larger, mostly {alpha}-helical COOH-terminal lobe with the nucleotide-binding region formed by the cleft between the two lobes (56). Generally, the kinase activation loop provides a mechanism to modulate kinase activity. In the unphosphorylated state, the activation loop tends to be structurally disordered, whereas following phosphorylation a well-ordered and "active" conformation of the loop is observed. Interestingly, the activation loop in both unphosphorylated and phosphorylated forms of ACK is highly ordered, suggesting that both ACK structures adopt the active conformation irrespective of phosphorylation state. Although this structural phenomenon is relatively novel, it is consistent with experimental data that show that the autophosphorylation of Tyr284 has a limited effect on ACK activity (22). The adenine moiety in ATP forms hydrogen bonds with the backbone atoms of the linker region residues Glu206 and Ala208 in the binding cleft (56). Such bonding patterns are observed for all known kinase inhibitors. The tyrosine kinase inhibitor PD158780 fits well in the active site of ACK and forms a hydrogen bond with Ala208 within the linker region (Fig. 6), thus providing mechanistic information in support of our in vitro studies that find PD158780 strongly inhibits kinase activity. We are currently studying additional kinase inhibitors and comparing their effect on ACK kinase activity, growth of v-Ha-Ras-transformed cells, and their hypothetical binding of ACK. The orientation of these ligands within ACK ATP-binding site reveals critical information about the structure of the active site that could be useful for the future de novo design of ACK-specific inhibitors.



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  		FIGURE 6. Visual representation of the three-dimensional structure of the kinase domain of ACK.A. {alpha} Carbons are depicted by a shaded ribbon with PD158780 in the binding pocket. B. Enlargement of ACK-PD158780 interaction. C. Structural drawing of PD158780.

 

   Materials and Methods
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
Acknowledgements
 References
 
Materials
Cellfectin was purchased from Invitrogen Life Technologies (Carlsbad, CA). Isopropyl-L-thio-B-D-galactopyranoside, glutathione, MBP, DTT, and anti-phosphotyrosine were purchased from Sigma (St. Louis, MO). Glutathione-Sepharose was purchased from Amersham Biosciences (Uppsala, Sweden). FITC-VAD-fmk was purchased from Promega (Madison, WI). The polyclonal antibodies for c-fos and ACK-1 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). [{gamma}-33P]ATP was purchased from NEN (Boston, MA). Ha-Ras antibodies and kinase inhibitors (PD158780, quercetin, wortmannin, PD157432, genistein, and radicicol) were purchased from Calbiochem (La Jolla, CA). V12Cdc42 and N17Cdc42 plasmids were generous gift from Dr. L-H. Wang (Mount Sinai School of Medicine, New York, NY).

Induction of c-fos Expression, Immunoprecipitation, and Western Blotting
NIH 3T3 cells (2.5 x 105 per 35 mm dish) were cultured in DMEM supplemented with 10% FCS. After overnight incubation, cells were transfected with vector pMV7 (control), pMV7-ACKKR, pMV7-ACKLF, or v-Ras cDNA. Other cells were cotransfected with pMV7-ACKKR and v-Ras cDNA using the Cellfectin reagent. Each plasmid (2.5 µg) was mixed with 10 µg Cellfectin and left for 20 minutes to form complexes. The cells were then incubated with the DNA:Cellfectin complex for 2 hours in serum-free medium. The medium was replaced with medium containing 10% FCS for an additional 2 hours. The cells were then collected and lysed in Laemmli SDS sample buffer. v-Ha-Ras and Cdc42 mutants were transfected using the same protocol, except that cells were incubated overnight after transfection.

For immunoprecipitation, cells (2 x 106 per sample) were lysed in a buffer containing 1% Triton X-100, 20 mmol/L HEPES (pH 7.4), 150 mmol/L NaCl, 10% glycerol, 1.5 mmol/L MgCl2, 1 mmol/L EGTA, 10 mmol/L sodium pyrophosphate, 0.2 mmol/L sodium orthovanadate, 50 mmol/L NaF, 0.5 mg/mL phenylmethylsulfonyl fluoride, and 0.5 µg/mL aprotinin. Each lysate was incubated with ACK-1 antibody (10 µg/sample) for 4 hours at 4°C. Protein A-Sepharose CL-4B (50 µL) was added to the lysate followed by additional incubation for 2 hours at 4°C. Sepharose beads were collected by centrifugation at 1,000 x g for 5 minutes (Eppendorf microfuge). The pellets were washed thrice with lysis buffer using the same protocol. Protein bound to Sepharose beads was recovered in Laemmli SDS sample buffer. Proteins were separated by SDS-PAGE and transferred to nylon membrane, and Western blotting was done according to the enhanced chemiluminescence protocol provided by the suppliers (Amersham Biosciences, Buckinghamshire, United Kingdom) using specific antibodies.

Plasmid Construction
A fragment of the ACK-1 gene (encoding amino acids 101-441) corresponding to the SH3 and kinase domains (named ACKD) was amplified by oligonucleotide-directed PCR using primers (5'-GAATTCTTTGAGTACGTCAAGAATGAG-3' and 5'-GAATTCTTAAAACGTGGGTCTGTCCTC-3'). The PCR product was digested with EcoRI and inserted into a bacterial expression vector, pGEX-2TH, using the EcoRI site. Accurate insertion of the PCR product was confirmed by nucleotide sequencing. Construction of the dominant-negative ACK mutant, ACK-1KR (K214R), has been described previously (24). The ACK-1KR insert was digested with restriction endonuclease and transferred into the mammalian expression vector pMV-7 (27).

Preparation of GST-ACK-1 Kinase Domain
Escherichia coli BL21 cells transformed with pGEX-ACKD were grown at 30°C to early logarithmic phase and protein expression was induced by adding 0.1 mmol/L isopropyl-L-thio-ß-D-galactopyranoside. After 3 hours of incubation, cells were harvested, resuspended in lysis buffer [50 mmol/L Tris (pH 7.5), 0.73 mol/L sucrose, 5 mmol/L MgCl2, 0.5% (v/v) NP40], and disrupted by sonication. Cells were centrifuged at 10,000 x g for 30 minutes at 4°C. The supernatant was applied to the glutathione-Sepharose column equilibrated with WED buffer [20 mmol/L Tris (pH 7.5), 2 mmol/L MgCl2, 1 mmol/L DTT] followed by washing with WED buffer. GST-ACKD was eluted with 5 mmol/L glutathione solution in 50 mmol/L Tris (pH 9.6). The eluate was dialyzed in WED buffer overnight and concentrated on a sucrose gradient. The expected size of the fusion protein (GST-ACKD) was confirmed by SDS-PAGE (data not shown), and the protein was used for kinase assays as described below.

Kinase Assay
The purified GST-ACKD (~5 µg per reaction) was incubated in kinase reaction buffer [50 mmol/L HEPES-KOH (pH 7.2), 10 mmol/L magnesium acetate, 5 mmol/L DTT] containing 7.5 µg MBP, 100 µmol/L ATP, and 4 µCi [{gamma}-33P]ATP for 10 minutes at 30°C. Reactions were stopped by addition of 5x Laemmli SDS sample buffer. Proteins were separated by SDS-PAGE, and radioactivity incorporated into the substrate was quantified by using the Kodak Imaging Station 2000R. For kinase inhibition experiments, GST-ACKD was preincubated with individual inhibitors in kinase buffer or kinase buffer alone (control) before the addition of MBP following the same protocol as described above. Experiments were done in triplicate.

Treatment of v-Ha-Ras-Transformed Cells with Kinase Inhibitors
To study the effects of ACK on cell proliferation, 2 x 104 cells per well were seeded into 24-well plates and cultured under standard cell culture conditions. After overnight culture, individual kinase inhibitors (at indicated concentration) or DMSO (control) were added to the culture. Cells were collected by trypsinization after 48 hours. Cell numbers were counted with a hemocytometer. To study ACK phosphorylation, cells were lysed with a buffer containing 1% Triton X-100, 20 mmol/L HEPES (pH 7.4), 150 mmol/L NaCl, 10% glycerol, 1.5 mmol/L MgCl2, 1 mmol/L EGTA, 10 mmol/L sodium pyrophosphate, 0.2 mmol/L sodium orthovanadate, 50 mmol/L NaF, 0.5 mg/mL phenylmethylsulfonyl fluoride, and 0.5 µg/mL aprotinin (11). Total protein (~1 mg) was used for immunoprecipitation of ACK-1.

ACK siRNA Treatment
A pair of cRNA primers of 21 nucleotides (Dharmacon Research, Inc.,Lafayette, Co.) corresponding to the 5' noncoding of the ACK-1 cDNA (5'-CAUUACCCGCCUAUCUCAUdTdT-3' and 5'-AUGAGAUAGGCGGGUAAUGdTdT-3') were annealed to form siRNA (a 19-nucleotide duplex stem with two-nucleotide overhangs on either side) according to the instruction provided by the manufacturer. v-Ha-Ras-transformed or parental NIH 3T3 cells were seeded into 6- or 24-well plates and incubated overnight. The annealed double-stranded ACK siRNA (0.16, 0.4, or 0.8 nmol/L in DMEM) or the sense strand oligonucleotide of ACK siRNA (0.8 nmol/L) was complexed with Cellfectin. siRNA:Cellfectin complexes were added to the serum-free medium and incubated for 3 hours. Cells were then replenished with medium containing 10% FCS and incubated for another 21 hours or as indicated elsewhere. Cells were collected and counted using a hemocytometer; alternatively, cell lysates were prepared for Western blotting. Western blotting was done using ACK-1 antibodies.

Analysis of Cell Cycle Arrest and Induction of Apoptosis
v-Ras-transformed cells (1 x 105) were seeded in a 35 mm dish and incubated under standard cell culture conditions overnight. Cells in DMEM were treated with Cellfectin, the sense strand of the siRNA:Cellfectin complex or the siRNA:Cellfectin complex for 3 hours. The medium was then replaced with DMEM containing 10% FCS and incubated for 21 hours at 37°C. Cells were harvested and used for Western blotting with specific antibodies or for cell cycle or caspase activation assays.

For cell cycle and caspase activation assays, cells were resuspended in PBS containing FITC-VAD-fmk for 10 minutes at room temperature. The cells were then fixed with ice-cold 70% ethanol for 30 minutes at 4°C. Following a rinse with PBS, the cells were resuspended in PBS containing RNase (0.1 mg/mL) and then stained with propidium iodine (10 µg/mL) for 10 minutes at room temperature. Cellular fluorescence from a sample of 15,000 cells was analyzed using a Coulter EPICS Profile II Flow Cytometer (Coulter Electronics, Miami, FL). Fluorescence excited at 488 nm was detected using a 525 ± 20 band pass filter. Histograms were analyzed using EPICS Workstation Software (version 4).

Nuclear DNA Fragmentation Assay
v-Ras-transformed cells (5 x 105) were seeded in 35 mm dishes and incubated overnight under standard cell culture conditions. Cells in DMEM were treated for 3 hours with Cellfectin, Cellfectin complexed with the sense strand of siRNA, Cellfectin complexed with the siRNA, or VP-16. The medium was replaced with DMEM containing 10% FCS and cells were incubated for 21 hours at 37°C. Cells were harvested and chromosomal DNA fragmentation was assayed using methods described previously (57).


   Acknowledgements
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
References
 
We thank Dr. L-H. Wang for kindly providing V12Cdc42 and N17Cdc42 plasmids.


   Notes
Top
 Notes
Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
Grant support: New Jersey Commission on Cancer Research grant 01-41-CCR-S-1 (A. Nur-E-Kamal) and NIH grant NS40394 (S. Meiners).

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.

4 Nur-E-Kamal et al., unpublished data. Back

Received 9/ 6/04; revised 3/13/05; accepted 3/18/05.


   References
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 

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