Interaction between c-Abl and Arg Tyrosine Kinases and Proteasome Subunit PSMA7 Regulates Proteasome Degradation

Molecular Cell 22, 317–327, May 5, 2006 ª2006 Elsevier Inc. DOI 10.1016/j.molcel.2006.04.007

Interaction between c-Abl and Arg Tyrosine Kinases and Proteasome Subunit PSMA7 Regulates Proteasome Degradation Xuan Liu,1 Wei Huang,1 Chufang Li,1 Ping Li,1 Jing Yuan,1 Xiaorong Li,1 Xiao-Bo Qiu,2 Qingjun Ma,1 and Cheng Cao1,* 1 Beijing Institute of Biotechnology Beijing 100850 China 2 Institute of Basic Medical Sciences Peking Union Medical College Beijing 100005 China

Summary Proteasome-mediated proteolysis is a primary protein degradation pathway in cells. The present study demonstrates that c-Abl and Arg (abl-related gene) tyrosine kinases associate with and phosphorylate the proteasome PSMA7 (a4) subunit at Tyr-153. Consequently, proteasome-dependent proteolysis is compromised. Notably, cells expressing a phosphorylation mutant of PSMA7(Y153F) display impaired G1/S transition and S/G2 progression, highlighting the biological significance of tyrosine phosphorylation of a proteasome subunit as an important cellular regulatory control. Introduction Proteasome-dependent proteolysis has emerged as a major regulatory mechanism that is broadly important during embryonic development, immune and stress responses, cell differentiation, cell cycle transitions, transcriptional regulation, and apoptosis (reviewed by Varshavsky [2005], Kloetzel [2004], Hershko [1997], and Varshavsky [2003]). Ubiquitination enzymes modify the cellular proteins for recognition by the proteasome. The proteasome core particle (20S proteasome) consists of four heterooligomeric ([a1-a7][b1-b7][b1-b7][a1-a7]) rings in which a subunits are required for proteasome assembly and 19S or 11S regulatory complex binding, and b subunits are responsible for protein degradation specified by their trypsin-, chymotrypsin-, and postglutamyl peptidyl hydrolytic activities (Coux et al., 1996; Voges et al., 1999; Glickman and Ciechanover, 2002). The nonreceptor protein tyrosine kinases c-Abl and Arg play important roles in regulating cell proliferation, apoptosis, adhesion, cell migration, and stress responses (Van Etten, 1999; Pendergast, 2002). The N-terminal portion of both c-Abl and Arg is composed of Src homology (SH) 3, SH2, and kinase domains (Goff et al., 1980; Kruh et al., 1990). The existence of C-terminal DNA binding motifs and nuclear localization signals in c-Abl enables shuttling between cytoplasmic and nuclear compartments (Van Etten et al., 1994; Kipreos and Wang, 1992), extending the exposure to additional Abl kinase substrates. Mice with targeted disruption of the c-abl gene are born runted, exhibit head and eye abnormalities, and *Correspondence: [email protected]

succumb as neonates with defective lymphopoiesis (Tybulewicz et al., 1991; Schwartzberg et al., 1991). Moreover, embryos deficient in both c-abl and arg exhibit defects in neurulation and die by 11 days postcoitus (Koleske et al., 1998). Other studies have demonstrated that, depending on its nuclear localization signals, c-Abl kinase function is associated with cell cycle arrest and DNA damage-induced apoptosis by interactions with p53 and its homolog, p73 (Goga et al., 1995; Yuan et al., 1996, 1999; Gong et al., 1999; Agami et al., 1999). c-Abl kinase is activated by several stimuli, including DNA damage, oxidative stress, Src tyrosine kinases, and growth factors (Baskaran et al., 1997; Sun et al., 2000; Plattner et al., 1999). For certain cellular regulatory proteins, c-Abl activity serves to stabilize protein levels. For example, c-Abl tyrosine kinase could stabilize p53 by blocking Mdm2-mediated p53 ubiquitination and nuclear export (Goldberg et al., 2002). As the homolog of p53, p73 is also stabilized by c-Abl (Tsai and Yuan, 2003). Moreover, proteasomal degradation of IkBa and ERKs was inhibited by c-Abl (Kawai et al., 2002; Buschbeck et al., 2005). Other studies have shown that activation of c-Abl by mutations or from its phosphorylation by Src family kinases results in decreased protein stability. This finding indicated that c-Abl could regulate its own degradation by a ubiquitin (ub)-proteasome pathway (Echarri and Pendergast, 2001). Activated cytoplamic c-Abl or Arg not only targets catalase for activation but also for destruction through the 26S proteasome, dictated by variations in cellular oxidative stress (Cao et al., 2003a). Thus, although there is evidence that Abl directs its substrate degradation through ub-proteasome pathway, the mechanism of degradation and the exact relationship between Abl and ub-proteasome pathway are unclear. The present study demonstrates that c-Abl and Arg physically interact with the proteasome PSMA7 subunit and that the tyrosine kinase activity of c-Abl and Arg is responsible for the Tyr-153 phosphorylation of PSMA7. The interaction and phosphorylation lead to an inhibition of proteasomal activity and cell cycle transition blocks. Results c-Abl and Arg Associate with PSMA7 We previously noted in a yeast two-hybrid analysis that c-Abl associates with the PSMA7 proteasome subunit (data not shown). To substantiate the c-Abl-PSMA7 and c-Abl-proteasome interaction, lysates from the human cell line 293T were subjected to immunoprecipitation with anti-c-Abl. By immunoblotting with an anti-PSMA7, anti-PSMA4, or anti-S5a (PSMD4, a 19S non-ATPase subunit) antibody, PSMA7 and the other two proteasome subunits were found in c-Abl immunoprecipitates (Figure 1A, left). To access the fractioned amount of free versus bound proteasomal subunits, the lysates were subjected to Superdex 200 gel filtration chromatography. Proteasome complexes were eluted at 14–16 ml fraction, and free PSMA7 was eluted at 24–26 ml fraction (Figure 1A, bottom right). Free

Molecular Cell 318

Figure 1. c-Abl and Arg Associate with PSMA7 (A) Left, lysates from 293T cells were subjected to immunoprecipitation with anti-c-Abl or IgG, fractionated by SDS-PAGE, and subsequently analyzed by immunoblotting with indicated antibodies. Right, lysates were fractionated by gel filtration chromatography, and selected fractions were blotted with antibodies as noted. Fractions 14–16 contain proteasomes, whereas fractions 24–26 contain free subunits. (B) Left, 293T cells were cotransfected with Myc-c-Abl and Flag-PSMA7 expression plasmids or Flag-vector, and anti-Flag or IgG immunoprecipitates were analyzed by immunoblotting with anti-Myc or anti-Flag antibody. Right, proteasome complexes were prepared from lysates of 293T cells cotransfected with Myc-c-Abl and Flag-PSMA7 expressing plasmids by gel filtration chromatography; the fraction 15 containing proteasome complexes was subjected to anti-Flag or IgG immunoprecipitation and analyzed by immunoblotting with indicated antibodies. (C) Left, 293T cells were cotransfected with Myc-PSMA7 and Flag-c-Abl or Flag-Arg expressing plasmids. The immunoprecipitates were analyzed by immunoblotting with anti-Myc or anti-Flag antibody as above. Right, the immunoprecipitates of Figure 1C were tested for proteasomal activity as follows. Flag peptide was used to elute Flag-c-Abl from the anti-Flag immunoprecipitates. The proteasomal peptidase substrate SucLeu-Leu-Val-Tyr-AMC was then incubated with the eluted protein. A 10% V/V lysate was employed as an activity control. The results are expressed as the mean 6 SD of three independent experiments. (D) Left, 293T cells were cotransfected with Flag-PSMA7 and Myc-c-Abl or Myc-c-Abl(K290R) expressing plasmids. The immunoprecipitates with anti-Flag or IgG were analyzed by immunoblotting with anti-Myc or anti-Flag antibody. Right, lysates of 293T cells transfected with Flagc-Abl or Flag-c-Abl(K290R) expressing plasmids were fractionated by gel filtration chromatography. Anti-Flag immunoprecipitates prepared from the fraction 15 were analyzed by immunoblotting with indicated antibodies.

PSMA7 and S5a were not detectable in the lysates of 293T cells (Figure 1A, right, top three panels). Therefore, c-Abl binds to PSMA7 in holoproteasome complexes. To confirm the binding of PSMA7 to c-Abl, 293T cells were cotransfected with plasmids expressing FlagPSMA7 and Myc-c-Abl. Immunoblot analysis of antiFlag immunoprecipitates with anti-Myc antibody showed a significant association between Flag-PSMA7 and Myc-c-Abl (Figure 1B, left). The same lysate was fractionated by gel filtration, and free form eluates relative to the proteasome form were accessed by Western blot with anti-Flag (Figure 1A, bottom right). A fraction containing proteasome complexes (fraction 15) was subjected to anti-Flag immunoprecipitation, and it was revealed by anti-Myc immunoblotting that Myc-c-Abl interacts with Flag-PSAM7 incorporated into proteasome complexes (Figure 1B, right). In addition, for cells coexpressing Flag-c-Abl and Myc-PSMA7, anti-Flag immu-

noprecipitates were found to coprecipitate the MycPSMA7 protein by anti-Myc immunoblotting (Figure 1C, left). In a similar coprecipitation experiment, the c-Abl-related Arg tyrosine kinase also forms complexes with PSMA7 (Figure 1C, left). Important to the potential function of these associations, it was observed that the Flag-c-Abl bound proteasomes had proteasomal peptidase activity (Figure 1C, right). To investigate whether the interaction was kinase activity dependent, lysates of 293T cells expressing Flag-PSMA7 and Mycc-Abl or kinase inactive Myc-c-Abl(K290R) were subjected to anti-Flag immunoprecipitation. Analysis of the precipitates demonstrated that, compared to wildtype c-Abl, Myc-c-Abl(K290R) formed less complexes with Flag-PSMA7 (Figure 1D, left). Further, lysates of 293T cells expressing Flag-c-Abl or Flag-c-Abl(K290R) were fractionated by gel filtration, and the proteasome-containing fraction was immunoprecipitated

Proteasome Regulation by Abl Tyrosine Kinases 319

tibody. The results showed that c-Abl binds to PSMA7 directly (Figure 2B). As control, Flag-c-Abl does not bind to IgG (Figure 2B, middle). Similar results were also obtained with Arg (data not shown). To define the interaction domain of c-Abl and Arg with PSMA7, lysates from 293T cells expressing Flag-PSMA7 were incubated with GST-c-Abl SH3, GST-c-Abl SH2 fusion proteins, or GST. Analysis of the absorbates by immunoblotting with anti-Flag demonstrated binding of PSMA7 to c-Abl SH3 and at a lesser extent to the c-Abl SH2 domain (Figure 2C). A similar experiment was performed to show the binding of PSMA7 to the SH3 and SH2 domains of Arg (Figure 2C). Consistent with these findings, we further observed that c-Abl SH3 coprecipitates with the 20S proteasome, containing PSMA7, PSMA2, PSMA4, and other components (Figure 2D). These findings confirm that a direct binding of PSMA7 in proteasomes occurs primarily through the SH3 and SH2 domains of c-Abl and Arg.

Figure 2. c-Abl and Arg Bind Directly to PSMA7 (A) Lysates from 293T cells transfected with Flag-c-Abl expressing plasmid were incubated with a GST or GST-PSMA7 fusion protein for 2 hr. The absorbates were analyzed by immunoblotting with anti-c-Abl (top). Loading of the GST proteins was assessed by Coomassie blue staining (bottom). (B) Anti-Flag or IgG immunoprecipitates prepared from cells transfected with Flag-c-Abl or Flag-vector expressing plasmids were subjected to SDS-PAGE and blotted onto nitrocellulose membrane. The nitrocellulose membrane was incubated with soluble GSTPSMA7 or IgG for 2 hr and then analyzed with anti-GST, anti-IgG, or anti-Flag antibody. (C) 293T cells were transfected with Flag-PSMA7 expressing plasmid. The GST fusion protein absorbates from cell lysates were analyzed by immunoblotting with anti-Flag antibody (top). (D) Purified 20S proteasomes were incubated with GST-c-Abl SH3 fusion proteins or GST proteins (as shown in Figure 2C, bottom). The absorbates were analyzed by immunoblotting with indicated antibodies.

with anti-Flag. Probing with proteasome subunit antibodies demonstrated that proteasome coprecipitated with Flag-c-Abl to a significantly greater extent than with Flag-c-Abl(K290R) (Figure 1D, right). These findings indicate that c-Abl and Arg associate with the PSMA7 in proteasome complexes in cells in a manner that is partly dependent on c-Abl kinase activity. Direct Binding of c-Abl and Arg to PSMA7 To further define the interaction between c-Abl and PSMA7, lysates from 293T cells were incubated with glutathione S-transferase (GST) or GST-PSMA7 fusion protein. It was demonstrated that c-Abl binds to GSTPSMA7, but not GST (Figure 2A). To rule out an indirect binding mediated by other components in the cell lysate, anti-Flag immunoprecipitates prepared from cells expressing Flag-c-Abl were subjected to SDS-PAGE and then blotted onto a nitrocellulose membrane. After incubation with soluble GST-PSMA7 fusion protein, the nitrocellulose membrane was treated with an anti-GST an-

c-Abl and Arg Phosphorylate PSMA7 on the Y153 Site The association between PSMA7 and c-Abl or Arg suggests that the proteasome subunit could be a substrate for these tyrosine kinases. This notion is supported by several lines of evidence. First, MCF-7 human tumor cell lysates were immunoprecipitated with anti-PSMA7 antibody and immunoblotted with an anti-phosphotyrosine (P-Tyr) antibody to demonstrate that PSMA7 was constitutively phosphorylated (Figure 3A, left). The phosphorylation was inhibited by pretreatment of cells with the c-Abl and Arg selective inhibitor STI571. Second, Flag-PSMA7 was coexpressed with c-Abl, and the proteasome complex fraction was immunoprecipitated with anti-Flag antibody. Immunoblotting with anti-P-Tyr antibody indicated that PSMA7 in proteasome complexes was phosphorylated (Figure 3A, right). Third, Flag-PSMA7 was coexpressed with c-Abl, Arg, kinase inactive c-Abl(K290R), or kinase inactive Arg(K337R) in c-abl2/2arg2/2 mouse embryo fibroblast (MEF) cells. Immunoblot analysis demonstrated that Flag-PSMA7 was phosphorylated by c-Abl and Arg, but not by cAbl(K290R) or Arg(K337R) (Figure 3B). In concert with these findings, treatment with STI571 inhibits PSMA7 phosphorylation mediated by endogenous c-Abl/Arg or by overexpressed Myc-c-Abl in 293T cells (Figure 3B). Whereas Flag-PSMA7 was expressed to comparable levels in the wild-type, c-abl2/2, or c-abl2/2arg2/2 MEFs, the PSMA7 protein was phosphorylated in wildtype MEFs, and in c-abl-deficient MEFs to a lesser extent, the phosphorylation was abrogated in c-abl2/2 arg2/2 MEFs (Figure 3C). Importantly, tyrosine phosphorylation of PSMA7 was restored upon Myc-c-Abl transfection into the c-abl2/2arg2/2 MEFs (Figure 3C). Finally, to obtain evidence of direct c-abl phosphorylation of PSMA7, recombinant GST-PSMA7 was incubated with Flag-c-Abl or Flag-Arg immunoprecipitates from transfected 293T cells in the presence of [g-32P]-ATP. Analysis of the reaction products by SDS-PAGE and autoradiography demonstrated phosphorylation of PSMA7 (Figure 3D and data not shown). Therefore, PSMA7 is a substrate of c-abl and Arg kinases in cells. We next examined whether phosphorylation of PSMA7 occurred in the context of the proteasome.

Molecular Cell 320

Figure 3. c-Abl and Arg Phosphorylate PSMA7 (A) Left, MCF-7 cells were treated with or without 10 mM STI571 for 12 hr. Anti-PSMA7 immunoprecipitates were analyzed by immunoblotting with anti-P-Tyr or anti-PSMA7 antibody. Right, lysates prepared from 293T cells cotransfected with Flag-PSMA7 and Myc-c-Abl expressing plasmids were subjected to gel filtration chromatography. Anti-Flag or IgG immunoprecipitates of the proteasome complexes fraction in the 15th elution volume were analyzed by immunoblotting with anti-P-Tyr or anti-Flag antibody. (B) c-abl2/2arg2/2 MEFs (left and middle) or 293T cells (right) were cotransfected with indicated vectors and treated with or without 10 mM STI571 for 12 hr. Anti-Flag immunoprecipitates were analyzed by immunoblotting with anti-P-Tyr or anti-Flag antibody. (C) MEFs, c-abl2/2 MEFs, or c-abl2/2arg2/2 MEFs were cotransfected with Flag-PSMA7 and Myc-c-Abl or pCMV-Myc expressing plasmids. Anti-Flag immunoprecipitates were analyzed by immunoblotting with anti-P-Tyr or anti-Flag antibody. (D) Recombinant GST-PSMA7 or GST-PSMA7 (Y153F) was incubated with purified Flag-c-Abl in the presence of [g-32P]-ATP. The reaction products were analyzed by SDS-PAGE and autoradiography. (E) Purified c-Abl (Upstate Biotechnology) was incubated with 26S proteasome for 30 or 90 min in the presence of ATP. The reaction products were analyzed by immunoblotting with anti-P-Tyr or anti-20S proteasome antibody. (F) The 90 min reaction products in (E) were separated by 2D electrophoresis, and the 2D gel was subjected to immunoblotting with anti-P-Tyr or anti-PSMA7 antibody, or analyzed by Coomassie Brilliant Blue staining. (G) 293T cells were cotransfected with indicated plasmids, and anti-Flag immunoprecipitates were analyzed by immunoblotting with anti-P-Tyr or anti-Flag antibody.

Proteasome Regulation by Abl Tyrosine Kinases 321

The 20S core subunit and the 19S regulatory subunit of the 26S proteasome were each phosphorylated by cAbl and Arg in vitro (Figure 3E and data not shown). Analysis by two-dimensional (2D) electrophoresis and anti-P-Tyr immunoblotting revealed that PSMA7 was the major tyrosine phosphorylated 20S subunit in 26S proteasome, whereas other subunits were found to be weakly phosphorylated (Figure 3F). These results demonstrate that PSMA7 in 26S proteasome can be specifically phosphorylated by c-Abl and Arg in vitro and in vivo. To determine the phosphorylation site(s) in PSMA7, recombinant GST-PSMA7 was incubated with c-Abl or Arg in the presence of [g-32P]-ATP and then subjected to tryptic digestion. Analysis of the radiolabeled peptides by protein sequencing indicated phosphorylation at tyrosine 153 (data not shown). Thus, PSMA7 Y153F was compared with wild-type PSMA7 for c-Abl- or Arg-mediated phosphorylation. GST-PSMA7 Y153F failed to be phosphorylated in vitro by c-Abl and Arg (Figure 3D and data not shown). To determine whether in vivo phosphorylation of PSMA7 is affected by the Y153F mutation, 293T cells were cotransfected with Flag-PSMA7 (or Flag-PSMA7[Y153F]) and Myc-c-Abl or Myc-Arg. Immunoblot analysis of anti-Flag immunoprecipitates with anti-P-Tyr showed the abrogation of tyrosine phosphorylation in PSMA7(Y153F) (Figure 3G). These findings indicate that c-Abl and Arg primarily phosphorylate PSMA7 at Tyr-153. c-Abl and Arg Inhibit Proteasome Activity In Vitro and In Vivo To investigate the effect of phosphorylation of PSMA7 by c-Abl and Arg on proteasome function, we monitored 20S proteasomal peptidase activity in the presence of c-Abl or Arg in vitro. It was found that degradation of the proteasome substrate Suc-LLVY-AMC by 20S (Figure 4A, left) as well as 26S (Figure 4A, right) proteasome was inhibited by c-Abl or Arg. Further, proteasome complexes in cells expressing Flag-c-Abl or FlagAbl(K290R) were prepared by gel filtration, and SucLLVY-AMC degradation was inhibited by c-Abl to a greater extent than by c-Abl(K290R) (Figure 4B, left). Moreover, increased proteasomal peptidase activity was observed in MCF-7 cells stably expressing a dominant-negative c-Abl(K290R) (Figure 4B, right). As another test, [35S] labeled proteins from c-abl2/2arg2/2 MEFs were incubated with 26S proteasomes in vitro, and the radioactivity of the acid soluble fraction (degraded products) was determined by liquid scintillation counting. Cotreatment with c-Abl caused 26S proteasome peptidase activity to be inhibited by more than 50%, approaching the maximal inhibition achieved with MG132, a known proteasomal peptidase inhibitor (Figure 4C). These experiments indicate that Abl and Arg phosphorylation is inhibitory to proteasomal activity. To determine whether an inability to phosphorylate PSMA7 may impact proteasomal peptidase activity, 293T cells were cotransfected with c-Abl and FlagPSMA7 or Flag-PSMA7(Y153F). The presence of the tagged PSMA7 and PSMA7(Y153F) in proteasome complexes was again demonstrated by gel filtration and immunoblotting (Figure 4D, bottom left). Therefore, Abl phosphorylation is not a prerequisite for proteasome

assembly. The in vivo degradation of short-lived proteins was assessed with a [35S]-methionine pulse-chase experiment for these transfected cells. Cells with PSMA7(Y153F)-loaded proteasomes had an increased overall proteasomal activity compared to vector or PMSA7-transfected cells (Figure 4D, left). Whereas vector and PSMA7-transfected cells showed elevated proteasomal degradation activity after STI571 treatment, there was no effect from the inhibitor in PSMA7(Y153F) cells (Figure 4D, left). Also, the c-abl2/2arg2/2 MEFs transfected with either Flag-PSMA7 or FlagPSMA7(Y153F) were subjected to the in vitro proteasomal peptidase activity assay, and no significant differences were revealed (Figure 4D, right). Overall, these findings indicate that tyrosine kinase activity on PSMA7 decreases the proteasomal activity. To further investigate the effect of c-Abl on in vivo degradation activity, we examined the stability of ZsGreen-ornithine decarboxylase (ODC), a fluorescenttagged protein containing a 50 aa 20S proteasome degradation domain of ODC. Because the cellular ZsGreen-ODC is highly susceptible to degradation by the proteasome, the protein will not accumulate normally in living cells (Li et al. [1998] and vector information from Clontech). MCF-7 cells stably transfected with ZsGreen-ODC were monitored by flow cytometry to assess the amount of the tagged protein. Therefore, a decrease in fluorescence intensity in this assay is indicative of an increased proteasomal activity. ZsGreenODC levels decreased significantly when the cells were treated with STI571 (Figures 4E, 5D, and 5F, right), indicating that an inhibition of c-Abl increases proteasomal degradation activity. Similar ZsGreen-ODC mRNA levels were detected by RT-PCR (data not shown). The dramatic decrease of ZsGreen-ODC protein level by the treatment of STI571 may reflect a modest decrease of proteasomal peptidase activity because the accumulation of ZsGreen-ODC is highly susceptible to proteasomal degradation. These data indicate that cellular proteasomal activity is at least partially inhibited by the c-Abl and Arg kinase activities. It is known that c-Abl regulates proteasomal degradation of p53 (Goldberg et al., 2002). Therefore, we tested whether recombinant Flag-p53 levels would be influenced by proteasomes loaded with Myc-PSMA7 or Myc-PSMA7(Y153F) in cotransfection and immunoblotting experiments. It was found that Flag-p53 levels were reduced with the cells expressing PSMA7(Y153F), and this effect was reversed by treatment with MG132 (Figure 4F). In sum, the kinase activity of c-Abl inhibits proteasomal degradation activity under physiological conditions and for specific proteasome substrates. Interaction of PSMA7 and c-Abl Is Regulated by Oxidative Stress and Ionizing Irradiation c-Abl is known to be activated by H2O2 (Sun et al., 2000) or ionizing radiation (Baskaran et al., 1997). To investigate whether the association of PSMA7 and c-Abl was regulated by oxidative stress, MCF-7 cells were treated with H2O2 and c-Abl-PSMA7 complexes were analyzed by coimmunoprecipitation. It was observed that treatment with 0.25 mM or 0.5 mM H2O2 resulted in increased formation of c-Abl-PSMA7 complexes (Figure 5A). The c-Abl-PSMA7 complexes were maximally observed for

Molecular Cell 322

Figure 4. Degradation Activity of the Proteasome Is Inhibited by c-Abl and Arg (A) Suc-Leu-Leu-Val-Tyr-AMC substrate was incubated with 20S (left) or 26S (right) proteasome in the presence of c-Abl, Arg, or imidazole eluates prepared from an uninfected Sf9 cell lysate for 1 hr, and the fluorescence signal of released AMC was measured. The effect of treatment with the proteasome inhibitor MG132 is also shown. The results are expressed as the mean 6 SD of three independent experiments. (B) Equivalent proteasome fractions (fraction 15 of gel filtration chromatography) from cells expressing Flag-c-Abl, Flag-c-Abl(K290R) (left), or MCF-7, MCF-7/c-Abl(K290R) (right) were monitored for proteasomal peptidase activity assayed as above. MG132 treatment is also shown. The results are expressed as the mean 6 SD of three independent experiments. (C) 26S proteasomes were incubated with c-Abl for 30 or 90 min in the presence of 2 mM ATP. Then [35S] labeled proteins prepared from MG132treated c-abl2/2arg2/2 MEFs were incubated with the pretreatment proteasome mix for 4 hr, and the acid soluble fraction was quantitated by liquid scintillation counting. The results are expressed as the mean 6 SD of three independent experiments. (D) Left, 293T cells expressing PSMA7 or PSMA7(Y153F) mutant were treated with (gray hatched boxes) or without 10 mM STI571 (open boxes) for 12 hr. After being pulsed with [35S]-methionine for 45 min, cells were washed and incubated with DMEM medium for 2 hr. Cell lysates together with medium were precipitated by 13% TCA, and the acid soluble fraction was quantitated by liquid scintillation counting (top). Also, proteasome fraction 15 was blotted with anti-Flag antibody (bottom). Right, normalized cell lysates of c-abl2/2arg2/2 MEFs transfected with Flag-PSMA7 or

Proteasome Regulation by Abl Tyrosine Kinases 323

Figure 5. Interaction of PSMA7 and c-Abl Is Regulated by Oxidative Stress and Ionizing Irradiation (A) MCF-7 cells were treated with the indicated concentrations of H2O2 for 1 hr. The anti-c-Abl immunoprecipitates were analyzed by immunoblotting with anti-PSMA7, anti-c-Abl, or anti-b-actin antibody. (B) MCF-7 cells were treated with 0.5 mM H2O2 for the indicated times. Anti-PSMA7 immunoprecipitates were analyzed by immunoblotting with the indicated antibodies. (C) 293T cells cotransfected with Myc-c-Abl and Flag-PSMA7 expressing plasmids were treated with indicated concentrations of H2O2 for 1 hr or with 0.5 mM H2O2 for indicated times. Anti-Flag immunoprecipitates were analyzed by immunoblotting with anti-P-Tyr or anti-Flag antibody. (D) MCF-7/ZsGreen-ODC cells were treated with or without 2.5 mM STI571 for 12 hr followed by treatment with the indicated concentration of H2O2 for 3 hr. The fluorescence intensity of cells was assayed by a fluorescence spectrophotometer. The results are expressed as the mean 6 SD of three independent experiments. As in flow cytometry, a reduction in the relative fluorescence units (RFU) is indicative of increased degradation of ZsGreen-ODC. (E) MCF-7 cells were treated with 0, 0.5, and 2 mM H2O2 for 3 hr in the presence of 0 or 10 mM MG132. Lysates were analyzed by immunoblotting with anti-c-Abl or anti-b-actin antibody. (F) Left, MCF-7 cells transfected with Flag-PSMA7 were treated with 10 Gy g-irradiation. Two hours after the treatment, anti-c-Abl immunoprecipitates prepared from lysates were analyzed by immunoblotting with indicated antibodies. Right, MCF-7/ZsGreen-ODC cells were treated with or without 2.5 mM STI571 for 12 hr followed by treatment with 10 Gy g-irradiation. The fluorescence intensity of cells was assayed by fluorescence spectrophotometer. The results are expressed as the mean 6 SD of three independent experiments.

the cells treated with 0.5 mM H2O2 for 0.5–3 hr (Figure 5B). Treatment with higher doses or longer times resulted in lower levels of c-Abl-PSMA7 complexes. Likewise, an increased tyrosine phosphorylation of PSMA7 was observed (Figure 5C). To further assess whether proteasomal activity was regulated by oxidative stress, MCF-7/ZsGreen-ODC cells were treated with HB2BOB2B in the presence of 0 or 2.5 mM STI571. Proteasomal activity was found to be inhibited by treatment with 0.25 mM or 0.5 mM H2O2, and STI571 treatment partially abrogated the effect (Figure 5D). Because it is known that c-Abl is degraded by ubiquitination via a proteasome pathway (Tybulewicz et al., 1991), we also examined c-Abl protein levels. Treatment with 0.5 mM H2O2, but not with 2 mM H2O2, resulted in an in-

creased c-Abl level in the cells (Figure 5E). By contrast, H2O2 treatment had little effect on c-Abl protein levels in the presence of the proteasome inhibitor MG132. Similar to the hydrogen peroxide treatment, c-AblPSMA7 association and PSMA7 phosphorylation were increased in MCF-7 cells treated with ionizing radiation (Figure 5F, left). Further, ionizing radiation caused a decrease in the in vitro proteasomal peptidase activity, and the resulting level of activity was significantly stimulated by STI571. (Figure 5F, right). These findings indicate that the interaction between c-Abl and PSMA7 is further induced when c-Abl is activated and that the proteasomal activity was inhibited by stimuli such as H2O2 and g-irradiation.

Flag-PSMA7(Y153F) expressing plasmids were subjected to proteasomal activity assay as described in (A). The results are expressed as the mean 6 SD of three independent experiments. (E) MCF-7/ZsGreen-ODC cells were treated with or without 2.5 mM STI571 for 12 hr, and cellular fluorescence intensity was evaluated by flow cytometry. (F) 293T cells cotransfected with indicated plasmids were treated with or without 10 mM MG132 for 2 hr, and cells lysates were analyzed by immunoblotting with anti-Flag or anti-b-actin antibody.

Molecular Cell 324

Figure 6. Expression of PSMA7 Y153F Mutant Induces a G1/S Cell Cycle Arrest (A) Lysates of G1/S-synchronized 293T cells transfected with PSMA7 or PSMA7 Y153F expressing plasmids were analyzed by immunoblotting with antibodies against the cell cycle transition mediator p27, cyclin A, and cyclin E. (B) 293T cells expressing PSMA7 or PSMA7 Y153F mutant were cultured to 80% confluence. Cell cycle was monitored by flow cytometry analysis after DNA staining with propidium iodide. The relative percentages of cells in G1, S, and G2 are as listed at the top of each panel. (C) 293T cells expressing PSMA7 or PSMA7 Y153F mutant were treated with 2 mM thymidine for G1/S synchronization. Cells were collected at the indicated time point after withdrawal of thymidine, and cell cycle was analyzed by flow cytometry. (D) The same experiment as in Figure 6C was carried out for c-abl2/2arg2/2 MEFs.

Phosphorylation of PSMA7 by c-Abl and Arg Regulates Cell Cycle Proteins important to cell cycle controls are frequently modulated by ubiquitin modification and the proteasome degradation pathway. As c-Abl and Arg are known to regulate cell cycle transitions in various contexts, we explored the significance of the c-Abl-PSMA7 association to cell cycle regulation via these mediators. Interest-

ingly, expression of the PSMA7(Y153F) in 293T cells results in a downregulation of cyclin A, cyclin E, and p27 levels (Figure 6A). Notably, expression of PSMA7(Y153F) was observed to induce a G1/S cell cycle arrest compared to wild-type PSMA7 expression (Figure 6B). To further investigate the cell cycle regulatory effect, cells were synchronized at G1/S by a thymidine block. Compared with vector and wild-type PSMA7 expressing

Proteasome Regulation by Abl Tyrosine Kinases 325

cells, PSMA7(Y153F) cells progress significantly slower from the G1/S transition into the cell cycle (Figure 6C). However, after synchronizing the PSMA7(Y153F) cells at G2/M with nocodozole, little effect on the dynamics of the G2/M to G1 transition was found (data not shown). As control, compared to Flag-PSMA7, PSMA7(Y153F) shows little if any effect on G1/S transition in MEFs deficient in both c-abl and arg, although a slightly earlier S/G2 transition was observed (Figure 6D). Discussion Role of PSMA7 and Interaction with c-Abl/Arg The PSMA7 subunit of 20S proteasome is located on the outer ring of the proteasome complex (Voges et al., 1999). Proteasomal activity is inhibited when PSMA7 associated with the X protein of Hepatitis B virus (HBX), a substrate of proteasome (Huang et al., 1996; Hu et al., 1999). Also, the hypoxia-inducible factor-1a (HIF-1a) physically interacts with PSMA7 and is targeted for proteasome-dependent degradation (Cho et al., 2001), suggesting an important regulatory mechanism in HIF-1a transactivation functions. The present studies demonstrate that Abl family nonreceptor tyrosine kinases regulate proteasome-mediated protein degradation by binding of SH3 and SH2 domains to PSMA7 subunit, and the association between c-Abl and PSMA7 is independent of c-Abl kinase activity (Figure 1D). Importantly, the present findings demonstrate that exposure of cells to 0.25 mM and 0.5 mM 0.5 H2O2 for 0.5–3 hr or to 10 Gy g-irradiation is associated with increased binding (Figures 5A, 5B, and 5F, left). Similar biphasic effects were observed for the formation of Abl-Arg and Abl-catalase complexes (Cao et al., 2003b). c-Abl and Arg Phosphorylate PSMA7 Phosphorylation of proteasome subunits has been demonstrated in several organisms. The phosphorylation of proteasomal a4 subunit at serine/threonine sites was demonstrated in yeast by proteomic approaches (Iwafune et al., 2002). Recent studies on phosphorylation of fish ooytes proteasomal a4 subunit by casein kinase Ia, a serine/threonine kinase, suggest that phosphorylation plays a role in the regulation of proteasomal activity during the meiotic cell cycle and, hence, is involved in fish maturation (Horiguchi et al., 2005). We are not aware of other studies demonstrating proteasome subunit tyrosine phosphorylation by a specific kinase. Our studies reveal that the human proteasomal subunit PSMA7 is a specific substrate of Abl tyrosine kinases. In addition, we have shown that c-Abl and Arg control the constitutive phosphorylation on PSMA7 Y153. The phosphotyrosine 153 moiety may facilitate PSMA7 and c-Abl/Arg interaction through the binding of c-Abl and Arg SH2 domains. Role of c-Abl and Arg in Cellular Proteasomal Activity and Cell Cycle The 20S and 26S proteasomal activities were inhibited by c-Abl and Arg binding and phosphorylation (Figure 4). Furthermore, c-Abl-PSMA7 complexes in proteasomes were increased by treatment with 0.25 and 0.5 mM H2O2 or 10 Gy g-irradiation, and cellular proteasomal activity was inhibited by the same treatments (Figure 5). Further, ubiquitination-proteasome-dependent degra-

Figure 7. A Model Depicting the Functional Consequences of c-AblMediated Phosphorylation on Proteasomal Activity

dation of endogenous c-Abl was blocked by treatment with 0.5 mM, but not 2 mM H2O2 (Figure 5E). Prior work indicated that H2O2 induced changes in proteasomal function (Grune, et al., 1995; Strack et al., 1996). Thus, our findings offer a molecular explanation for the previous data by implicating the Abl family kinases in terms of their binding associations, protein levels, and activities with the proteasomal peptidase mechanism. Proteasomal activity was upregulated by the expression of the PSMA7(Y153F) protein incapable of being phosphorylated by c-Abl or Arg (Figure 4D, left). Consequently, we observed that the levels of p53, p27, cyclin A, and cyclin E were downregulated by the effect of the mutant protein (Figures 4F and 6A). These results and our other findings further delineate how c-Abl and Arg are able to play important roles at multiple cell cycle transition stages (Figures 6B and 6C) and by a common mechanism (Figure 7). In all likelihood, the upregulation of p27 by c-Abl may also contribute to the c-Abl-dependent G0/G1 cell cycle arrest induced by g-irradiation (Yuan et al., 1996). Therefore, it is likely that c-Abl and Arg activities on the proteasomal function in the ordinary cell cycle transition and serve as a regulatory node during circumstances of oxidative or genotoxic stress. It is intriguing that c-Abl (Arg) regulates the activity of proteasome and, as a result, regulates cell cycle progression. Therefore, at least some of the defects associated with c-abl and arg deficiency may be attributable to changes in proteasomal function. c-abl2/2, arg2/2, or cabl2/2arg2/2 mice progress incompletely through embryonic development where there is an essential, formative role for the integrity of cell cycles. Potentially, the cause of massive embryonic abnormalities may result from a failure of proteasome downmodulation. In summary, the significance of phosphotyrosine modification of PSMA7 toward control of proteasomal activity has been identified. The negative regulation imparted by Abl family nonreceptor tyrosine kinases plays an important role in cell cycle progression by controlling the ub-proteasome pathway. It is expected that other factors that influence c-Abl and Arg activity may signify additional means to control proteasomal function. This study contributes to the growing body of knowledge of how these important tyrosine kinases represent a necessary nodal point toward integrating the diversity of positive and negative regulatory signals of cell homeostasis. Experimental Procedures Cell Culture and Transfections 293T, MCF-7, MCF-7/ZsGreen-ODC, and MEFs derived from wildtype, c-abl2/2, and c-abl2/2arg2/2 littermates (Koleske et al.,

Molecular Cell 326

1998), were grown in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (Hyclone), 2 mM L-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin. K562 cells were grown in RPMI Medium 1640 (GiBCO). Cells were treated with STI571 (Novartis) or MG132 (Sigma) as noted in the text. Transient transfections were performed with LipofectAMINE 2000 (Invitrogen). Vectors and Epitope Tagging of Proteins Flag-tagged c-Abl, Arg, and PSMA7 and their mutants were expressed by cloning the genes into the pcDNA3-based Flag vector (Invitrogen). Myc-tagged c-Abl, Arg, and PSMA7 vector were prepared by cloning into pCMV-Myc (Clontech). Glutathione Stransferase (GST) fusion proteins were generated by expression in pGEX4T-2-based vectors (Amersham Biosciences Biotech, Inc) in Escherichia coli BL21 (DE3). Immunoprecipitation and Immunoblot Analysis Cell lysates were prepared in lysis buffer (50 mM Tris-HCl [pH 7.5], 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 10 mM sodium fluoride, 10 mg/ml aprotinin, 10 mg/ml leupeptin, and 10 mg/ml pepstatin A) containing 1% Nonidet P-40. Soluble proteins were subjected to immunoprecipitation with anti-Flag (M2, Sigma), anti-Myc (Santa Cruz), anti-c-Abl (K-12, Santa Cruz), anti-PSMA7 (Affinity, Exeter UK), or anti-mouse IgG antibody (Sigma). An aliquot of the total lysate (5%, v/v) was included as a control. Immunoblot analysis was performed with anti-Myc, HRP-conjugated anti-Flag (Sigma), anti-c-Abl (Santa Cruz), anti-b-actin (Sigma), HRP-conjugated antiP-Tyr (Upstate Biotechnology), anti-p27 (Santa Cruz), anti-CyclinA (Santa Cruz), anti-cyclin E (Santa Cruz), anti-PSMA7, anti-PSMA2 (Affinity, Exeter UK), anti-PSMA4 (Affinity, Exeter UK), or anti-S5a (Affinity, Exeter UK) antibody. The antigen-antibody complexes were visualized by chemiluminescence (PerkinElmer Life Sciences). Protein Binding Assays In GST pull-down experiments, cell lysates were incubated for 2 hr at 4ºC with 5 mg purified GST or GST fusion proteins bound to glutathione beads. The adsorbates were washed with lysis buffer and then subjected to SDS-PAGE and immunoblot analysis. An aliquot of the total lysate (5%, v/v) was included as a loading control on the SDS-PAGE. In direct binding assays, immunoprecipitates were separated by SDS-PAGE and then blotted onto nitrocellulose membranes. Membranes were subsequently incubated with purified GST-fusion proteins for 2 hr at room temperature. The GST fusion proteins binding to nitrocellulose were probed with anti-GST antibody. Proteasome Isolation by Gel Filtration Chromatography Proteasome complexes were separated from free proteasome subunits by gel filtration with Superdex 200 gel packed in a 10 3 500 mm SR10/50J column. One milliliter cell lysate was loaded and eluted with 50 mM Tris-HCl (pH 8.0) containing 0.1 M NaCl and 2 mM ATP. Fractions of 1 ml were collected with a FC900 fraction collector. Proteasome complexes eluted at an elution volume (Ve) of 15 ml, whereas free PSMA7 eluted at 25 ml. Kinase Assays Purified GST-PSMA7 (2 mg) was incubated with Abl expressed in E. coli (Biolabs), His-tagged Arg, and c-Abl expressed by baculoviral vector in Sf9 insect cells (Upstate Biotechnology) in kinase buffer (20 mM HEPES [pH 7.5], 75 mM KCl, 10 mM MgCl2, and 10 mM MnCl2) containing 2.5 mCi of [g-32P]-ATP for 30 min at 37ºC. The reaction products were analyzed by SDS-PAGE and autoradiography. Sf9 cells expressing baculovirus-produced c-Abl or Arg were the source of c-Abl and Arg proteins by Nickel chelating absorption and imidazole elution methods. Imidazole eluates of Nickel chelating resin adsorbates of an uninfected Sf9 cell lysate were employed as negative control. 2D Gel Electrophoresis Purified 26S proteasomes were resuspended in the rehydration buffer (9 M urea, 2.2% CHAPS, 65 mM dithiothreitol, and 0.3% Immobilone pH gradient (IPG) buffer). The 7-cm pH 3-10 NL (nonlinear) IPG strip (Amersham Biosciences) was rehydrated with sample buffer. The loaded sample was focused for 30 min at 200 V, 30 min

at 500 V, and 1 hr at 2000 V followed by 8000 V for a total of 90 kVh, and then fractionated by 12.5% SDS-PAGE. Spots were visualized after immunoblotting or by Coomassie Brilliant Blue staining. Proteasomal Activity Assay Proteasomal peptidase activity was assayed by incubating AMCconjugated peptide substrates (Succinyl-leucyl-leucyl-valyl-tyrosyl7-amino-4-methylcoumarin, CalBiochem) with proteasome (Affinity, Exeter UK) for 1 hr at 37ºC in reaction buffer (25 mM HEPES, 0.5 mM EDTA [pH 7.6], and 0.3% SDS). The relative fluorescence was assayed at an excitation wavelength of 380 nm and an emission wavelength of 460 nm by fluorescence spectrometer. The relative fluorescence intensity is calculated by the mean 6 SD of three experiments. To assess ub-dependent proteolysis of 26S proteasome, c-abl2/2 arg2/2 MEFs were pulsed with [35S]-methionine for 45 min in the presence of 10 mM MG132. The lysates containing labeled short-lived proteins were passed through Econo-Pac 10DG columns (Bio-Rad) to remove unincorporated [35S]-methionine and MG132. c-Abl kinase treated or untreated 26S proteasome (Affinity, Exeter UK) was incubated with purified [35S]-methionine-labeled short-lived proteins in energy buffer (5 mM MgCl2 and 2 mM ATP) at 37ºC for 4 hr. The acid-soluble radioactivity was measured as an indicator of the 26S proteasome ability to degrade ubiquitinated short-lived proteins. The average of the CPM value from triplicated experiments was used to calculate the proteasomal degradation activity. In Vivo Proteasomal Activity Assay MCF-7/ZsGreen-ODC cells stably express the ZsGreen-ODC fusion proteins (Clontech) were treated with STI571, H2O2, or ionizing radiation (IR). Fluorescence of ZsGreen-ODC was measured at an excitation wavelength of 496 nm and an emission wavelength of 506 nm by flow cytometry (BD Biosciences). Treatments were in triplicate, and the average of the geometric means of the fluorescence intensity obtained from 104 cells of each sample was used to calculate the proteasomal degradation activity of the cells. To assess ub-dependent proteolysis of 26S proteasome in vivo, 293T cells expressing PSMA7 or PSMA7 Y153F mutant were pulsed with 10 Ci/mL [35S]-methionine to label short-lived proteins. After 45 min, cells were washed and fed in normal DMEM medium, and cells together with medium were collected at 0 and 2 hr. After cell lysis, 13% TCA was used to sediment the undegraded proteins and the supernatant was subjected to liquid scintillation counting for quantitation. The average of the CPM value obtained from triplicated experiments was used to calculate the proteasomal degradation activity. Cell Cycle Synchronization and Cell Cycle Assay To synchronize cells at the G1/S boundary, 293T cells were treated at 50% confluence with 2 mM thymidine (Sigma). After 12 hr, cells were washed and cultured with fresh medium for 8 hr before the addition of 2 mM thymidine to treat for another 12 hr. To synchronize cells at the G2/M boundary, cells were treated with 20 ng/ml nocodazole (Sigma) for 12 hr. After the release of the thymidine or nocodazole block by placing cells in fresh medium, the cell cycle was analyzed by flow cytometry analysis after staining with propidium iodide. The percentage of cells with G1, S, or G2 DNA is presented as the mean of three independent experiments in round figures. Acknowledgments This investigation was supported by grants 30270316 and 30370739 and Distinguished Young Scholars Grant 30525033 awarded by the Natural Science Foundation of China and also supported by grant 2005AA218080 awarded by National ‘‘863’’ HighTech Development Program. The authors acknowledge Donald Kufe (Dana Farber Cancer Institute) for insightful discussion and Dr. Tony Koleske (Yale University) for providing the c-abl2/2 and c-abl2/2arg2/2 MEFs. The authors also acknowledge David Weaver, Zhimin Yuan, and Rebecca Chakraborty for their great help in preparation of the manuscript. Received: April 17, 2005 Revised: September 7, 2005 Accepted: April 10, 2006 Published: May 4, 2006

Proteasome Regulation by Abl Tyrosine Kinases 327

References

Kipreos, E.T., and Wang, J.Y.J. (1992). Cell cycle-regulated binding of c-Abl tyrosine kinase to DNA. Science 256, 382–385.

Agami, R., Blandino, G., Oren, M., and Shaul, Y. (1999). Interaction of c-Abl and p73a and their collaboration to induce apoptosis. Nature 399, 809–813.

Kloetzel, P.M. (2004). Generation of major histocompatibility complex class I antigens: functional interplay between proteasomes and TPPII. Nat. Immunol. 5, 661–669.

Baskaran, R., Wood, L.D., Whitaker, L.L., Canman, C.E., Morgan, S.E., Xu, Y., Barlow, C., Baltimore, D., Wynshaw-Boris, A., Kastan, M.B., and Wang, J.Y. (1997). Ataxia telangiectasia mutant protein activates c-Abl tyroaine kinase in response to ionizing radiation. Nature 387, 516–519.

Koleske, A.J., Gifford, A.M., Scott, M.L., Nee, M., Bronson, R.T., Miczek, K.A., and Baltimore, D. (1998). Essential roles for the Abl and Arg tyrosine kinases in neurulation. Neuron 21, 1259–1272.

Buschbeck, M., Hofbauer, S., Croce, L., Keri, G., and Ullrich, A. (2005). Abl-kinase-sensitive levels of ERK5 and its intrinsic basal activity contribute to leukaemia cell survival. EMBO Rep. 6, 63–69. Cao, C., Leng, Y., Liu, X., Yi, Y., Li, P., and Kufe, D. (2003a). Catalse is regulated by ubiquitination and proteosomal degradation. Role of the c-Abl and Arg tyrosine kinases. Biochemistry 42, 10348–10353. Cao, C., Leng, Y., Li, C., and Kufe, D. (2003b). Functional interaction between the c-Abl and Arg protein-tyrosinekinase in the oxidative stress response. J. Biol. Chem. 278, 12961–12967. Cho, S., Choi, Y., Kim, J., Jeong, S., Kim, J., Kim, S., and Ryu, S. (2001). Binding and regulation of HIF-1a by a subunit of the proteasome complex, PSMA7. FEBS Lett. 498, 62–66.

Kruh, G.D., Perego, R., Miki, T., and Aaronson, S.S. (1990). The complete coding sequence of arg defines the Abelson subfamily of cytoplasmic tyrosine kinases. Proc. Natl. Acad. Sci. USA 87, 5802–5806. Li, X., Zhao, X., Fang, Y., Jiang, X., Duong, T., Fan, C., Huang, C.C., and Kain, S.R. (1998). Generation of destabilized green fluorescent protein as a transcription reporter. J. Biol. Chem. 273, 34970–34975. Pendergast, A.M. (2002). The Abl family kinases: mechanisms of regulation and signaling. Adv. Cancer Res. 85, 51–100. Plattner, R., Kadlec, L., DeMali, K.A., Kazlauskas, A., and Pendergast, A.M. (1999). c-Abl is activated by growth factors and Src family kinases and has a role in the cellular response to PDGF. Genes Dev. 13, 2400–2411.

Coux, O., Tanaka, K., and Goldberg, A.L. (1996). Structure and functions of the 20S and 26S proteasomes. Annu. Rev. Biochem. 65, 801–847.

Schwartzberg, P.L., Stall, A.M., Hardin, J.D., Bowdish, K.S., Humaran, T., Boast, S., Harbison, M.L., Robertson, E.J., and Goff, S.P. (1991). Mice homozygous for the ablm1 mutation show poor viability and depletion of selected B and T cell populations. Cell 65, 1165–1175.

Echarri, A., and Pendergast, A.M. (2001). Activated c-Abl is degraded by the ubiquitin-dependent proteasome pathway. Curr. Biol. 11, 1759–1765.

Strack, P.R., Waxman, L., and Fagan, J.M. (1996). Activation of the multicatalytic endopeptidase by oxidants. Effects on enzyme structure. Biochemistry 35, 7142–7149.

Glickman, M.H., and Ciechanover, A. (2002). The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction. Physiol. Rev. 82, 373–428.

Sun, X., Wu, F., Datta, R., Kharbanda, S., and Kufe, D. (2000). Interaction between protein kinase C d and the c-Abl tyrosine kinase in the cellular response to oxidative stress. J. Biol. Chem. 275, 7470– 7473.

Goff, S., Gilboa, E., Witte, O., and Baltimore, D. (1980). Structure of the Abelson murine leukemia virus genome and the homologous cellular gene Studies with cloned viral DNA. Cell 22, 777–785. Goga, A., Liu, X., Hambuch, T.M., Senechal, K., Major, E., Berk, A.J., Witte, O.N., and Sawyers, C.L. (1995). p53 dependent growth suppression by the c-Abl nuclear tyrosine kinase. Oncogene 11, 791– 799. Goldberg, Z., Vogt Sionov, R., Berger, M., Zwang, Y., Perets, R., Van Etten, R.A., Oren, M., Taya, Y., and Haupt, Y. (2002). Tyrosine phosphorylation of Mdm2 by c-Abl: implications for p53 regulation. EMBO J. 21, 3715–3727. Gong, J., Costanzo, A., Yang, H., Melino, G., Kaelin, W., Jr., Levrero, M., and Wang, J.Y.J. (1999). The tyrosine kinase c-Abl regulates p73 in apoptotic response to cisplatin-induced DNA damage. Nature 399, 806–809. Grune, T., Reinheckel, T., Joshi, M., and Davis, K.J. (1995). Proteolysis in cultured liver epithelial cells during oxidative stress. Role of the multicatalytic proteinase complex, proteasome. J. Biol. Chem. 270, 2344–2351. Hershko, A. (1997). Role of ubiquitin-mediated proteolysis in cel cycle control. Curr. Opin. Cell Biol. 9, 788–799. Horiguchi, R., Yoshikuni, M., Tokumoto, M., Nagahama, Y., and Tokumoto, T. (2005). Identification of a protein kinase which phosphorylates a subunit of the 26S proteasome and changes in its activity during meiotic cell cycle in goldfish oocytes. Cell. Signal. 17, 205– 215. Hu, Z., Zhang, Z., Doo, E., Coux, O., Goldberg, A.L., and Liang, T.J. (1999). Hepatitis B virus X protein is both a substrate and a potential inhibitor of the proteasome comoplex. J. Virol. 73, 7231–7240. Huang, J., Kwong, J., Sun, E.C.-Y., and Liang, T.J. (1996). Proteasome complex as a potential cellular target of hepatitis B virus X protein. J. Virol. 70, 5582–5591. Iwafune, Y., Kawasaki, H., and Hirano, H. (2002). Electrpohoretic analysis of phosphorylation of the yeast 20S proteasome. Electrophoresis 23, 329–338. Kawai, H., Nie, L., and Yuan, Z.M. (2002). Inactivation of NF-kappaBdependent cell survival, a novel mechanism for the proapoptotic function of c-Abl. Mol. Cell. Biol. 22, 6079–6088.

Tsai, K.K., and Yuan, Z.M. (2003). c-Abl stabilizes p73 by a phosphorylation-augmented interaction. Cancer Res. 63, 3418–3424. Tybulewicz, V.L.J., Crawford, C.E., Jackson, P.K., Bronson, R.T., and Mulligan, R.C. (1991). Neuonatal lethality and lymphopenia in mice with a homozygous disruption of the c-abl proto-oncogene. Cell 65, 1153–1163. Van Etten, R.A. (1999). Cycling, stressed out and nervous: Cellular functions of c-Abl. Trends Cell Biol. 9, 179–186. Van Etten, R.A., Jackson, P.K., Baltimore, D., Standers, M.C., Matsuddaira, P.T., and Janmey, P.A. (1994). The COOH terminus of the c-Abl tyrosine kinase contains distinct F- and G-Actin binding domains with bundling activity. J. Cell Biol. 124, 325–340. Varshavsky, A. (2003). The N-end rule and regulation of apoptosis. Nat. Cell Biol. 5, 373–376. Varshavsky, A. (2005). Regulated protein degradation. Trends Biochem. Sci. 30, 283–286. Voges, D., Zwickl, P., and Baumeister, W. (1999). The 26S proteasome: a molecular machine designed for controlled proteolysis. Annu. Rev. Biochem. 68, 1051–1068. Yuan, Z., Huang, Y., Whang, Y., Sawyers, C., Weichselbaum, R., Kharbanda, S., and Kufe, D. (1996). Role for c-Abl tyrosine kinase in growth arrest response to DNA damage. Nature 382, 272–274. Yuan, Z.M., Shioya, H., Ishiko, T., Sun, X., Huang, Y., Lu, H., Kharbanda, S., Weichselbaum, R., and Kufe, D. (1999). p73 is regulated by tyrosine kinase c-Abl in the apoptotic response to DNA damage. Nature 399, 814–817.