Regulation of p73 by Hck through kinase-dependent and independent mechanisms
© Paliwal et al; licensee BioMed Central Ltd. 2007
Received: 28 November 2006
Accepted: 30 May 2007
Published: 30 May 2007
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© Paliwal et al; licensee BioMed Central Ltd. 2007
Received: 28 November 2006
Accepted: 30 May 2007
Published: 30 May 2007
p73, a p53 family member is a transcription factor that plays a role in cell cycle, differentiation and apoptosis. p73 is regulated through post translational modifications and protein interactions. c-Abl is the only known tyrosine kinase that phosphorylates and activates p73. Here we have analyzed the role of Src family kinases, which are involved in diverse signaling pathways, in regulating p73.
Exogenously expressed as well as cellular Hck and p73 interact in vivo. In vitro binding assays show that SH3 domain of Hck interacts with p73. Co-expression of p73 with Hck or c-Src in mammalian cells resulted in tyrosine phosphorylation of p73. Using site directed mutational analysis, we determined that Tyr-28 was the major site of phosphorylation by Hck and c-Src, unlike c-Abl which phosphorylates Tyr-99. In a kinase dependent manner, Hck co-expression resulted in stabilization of p73 protein in the cytoplasm. Activation of Hck in HL-60 cells resulted in tyrosine phosphorylation of endogenous p73. Both exogenous and endogenous Hck localize to the nuclear as well as cytoplasmic compartment, just as does p73. Ectopically expressed Hck repressed the transcriptional activity of p73 as determined by promoter assays and semi-quantitative RT-PCR analysis of the p73 target, Ipaf and MDM2. SH3 domain- dependent function of Hck was required for its effect on p73 activity, which was also reflected in its ability to inhibit p73-mediated apoptosis. We also show that Hck interacts with Yes associated protein (YAP), a transcriptional co-activator of p73, and shRNA mediated knockdown of YAP protein reduces p73 induced Ipaf promoter activation.
We have identified p73 as a novel substrate and interacting partner of Hck and show that it regulates p73 through mechanisms that are dependent on either catalytic activity or protein interaction domains. Hck-SH3 domain-mediated interactions play an important role in the inhibition of p73-dependent transcriptional activation of a target gene, Ipaf, as well as apoptosis.
p73 is a transcription factor that shares significant homology with the tumor suppressor protein p53 and mediates cellular functions such as cell cycle arrest, differentiation, senescence and apoptosis [1, 2]. Alternate promoter usage and splicing give rise to p73 variants differing at the N and C-terminus, p73 α and β forms being the predominant transactivation competent forms [3, 4]. p73 and p53 bind common response elements and transactivate an overlapping set of target genes, though with differing efficiencies [1, 5–7]. p73 knockout mice exhibit severe neurological, inflammatory and pheromonal defects but lack spontaneous tumor formation . Forced expression of p73 activates target genes like p21, Bax, IGF BP3, CyclinG, Mdm2, caspase-1, etc and promotes apoptosis in both p53 positive as well as negative cell lines [1, 5–7, 9].
Cellular p73 protein is maintained at very low levels and activation is controlled at transcriptional as well as post-translational levels. p73 is induced in response to a range of DNA damaging agents as well as during differentiation of many cell types . Multiple mechanisms like post-translational modifications, protein-protein interaction and sub-cellular compartmentalization regulate transactivation potential and apoptosis inducing property of p73 [10, 11]. Several viral and cellular proteins interact with p73 and regulate its activity . While some of the interacting proteins enhance transactivation potential of p73 by stabilizing it [12–14], others inhibit transactivation property of p73 [15–20]. p73 is also known to interact with and cooperate with other transcription factors to regulate gene expression [9, 21]. Interaction with c-Abl through SH3 domain in response to DNA damage results in phosphorylation at Tyr99, stabilization and activation [12, 22, 23]. c-Abl can also activate p38 MAP kinase to phosphorylate p73 leading to its stabilization .
Src family kinases (SFKs) are non receptor tyrosine kinases involved in the regulation of diverse cellular functions like proliferation, differentiation, survival, adhesion, motility and angiogenesis . Apart from a catalytic domain, they possess SH2, SH3 and SH4 domains required for sub-cellular targeting and protein interaction and therefore have cellular functions dependent on catalytic activity as well as protein interaction domains [26, 27]. Hck is a SFK showing restricted expression in hematopoietic cells of myeloid and monocytic lineage and in embryonic stem cells [28–30]. It exists as two isoforms (p61 and p59 in humans), which arise due to alternate translational start sites . Hck is activated in response to cytokines and is an important component of signaling pathways in activated macrophages [32–34]. Hck levels increase during differentiation of monocytes and it plays a role in phagocytosis, adhesion, respiratory burst, granule secretion and apoptosis [35–37]. Several proteins like Cbl, Stat-3, WASP, WIP, ELMO, ACK1 and C3G interact with Hck through its SH3 domain [36–40]
The mechanisms that regulate stability and pro-apoptotic activity of p73 are distinct from those used by p53 and regulation by tyrosine phosphorylation has been shown only for p73 . Though initial studies described Tyr-99 as the major site of phosphorylation by cAbl, it has been shown that several other tyrosine residues are also targeted . But thus far, no other tyrosine kinase has been described to phosphorylate or regulate p73 activity. Hck interacts with c-Abl and they modulate each other's activity . Given the role of SFKs in mediating survival and apoptotic pathways in cells, we investigated the role of Hck in regulating p73 activity and function. Our results identify p73 as a novel interacting partner and substrate of Hck, and that Hck regulates p73 through kinase dependent as well as independent functions. We also provide evidence for functional interaction between p73 and Hck leading to regulation of p73 induced apoptosis.
Treatment of myelomonocytic cell lines with mercuric chloride (HgCl2) has been shown to specifically activate Hck, and has been used to identify substrates of Hck [36, 45, 46]. Treatment of HL-60 cells with DMSO showed an increase in protein levels of both p73α and Hck (Fig. 2D). To find out whether endogenous p73 gets tyrosine phosphorylated upon activation of Hck, HL-60 cells were differentiated for 48 hours with DMSO and then subjected to HgCl2 treatment. The lysates were immunoprecipitated with p73 and control antibody. It was observed that endogenous p73 gets tyrosine phosphorylated upon activation of Hck as determined by western blotting with phosphotyrosine antibody (Fig. 2E, right panel). The left panel of Fig. 2E shows the western blots of whole cell lysate (WCL) with phosphotyrosine, p73 and Hck antibodies.
Unlike other Src family kinases such as Src, Lyn and Fyn [51–55], no evidence is available for nuclear localization of Hck. It was therefore important to evaluate that presence of Hck in the nucleus is not a feature of exogenously expressed protein. We determined the sub-cellular distribution of endogenous Hck in a myeloid cell line HL-60. As shown in Fig. 5B, endogenous Hck distributed to both nuclear and cytosolic compartments as evidenced by sub-cellular fractionation. To confirm that sub cellular fractions were not contaminated, the fractions were analysed by western blotting for Calnexin (an ER protein, present in post nuclear fraction) and PARP (present only in nuclear fraction). The presence of endogenous Hck in the nucleus was confirmed by indirect immunofluorescence assay. HL-60 cells were differentiated with 10 ng/ml of 12-O-tetradecanoylphorbol-13-acetate (TPA), which allows cells to adhere to the coverslips. After 24 hours, cells were fixed and immunostained with Hck antibody and analysed by confocal microscopy. The middle section passing through the nucleus was analysed for determination of Hck localization. As shown in Fig. 5C, endogenous Hck localized to the nucleus. The cells were also stained without primary antibody for control.
Since Hck inhibited the transcriptional activity of p73α isoform in a kinase-independent manner, we explored the possibility of the involvement of SH3 domain. Towards this end, a critical tryptophan was mutated to alanine (W93A). This mutation in Hck abolished the binding of SH3 domain to proline rich sequences of proteins as well as enhanced its kinase activity . W93A-Hck (mSH3-Hck) was transfected in the presence and absence of p73α along with Ipaf-CAT reporter construct. mSH3-Hck was not able to inhibit p73α mediated transcriptional activity of Ipaf promoter whereas WT-Hck showed inhibition (Fig. 6D), suggesting that this inhibition is dependent on functional SH3 domain of Hck. To analyse if this property was unique to Hck, the effect of Src on Ipaf-promoter transactivation was examined. HeLa cells were transfected with p73α (2 ng) and c-Src or Hck (200 ng each) along with Ipaf-CAT promoter (200 ng) and cell lysates subjected to reporter activity assay. c-Src inhibited p73α mediated transactivation by 26.21% as compared to Hck which inhibited the activity by 60% (Fig. 6E). These results suggest that Hck is a more potent inhibitor of p73 induced Ipaf-promoter transactivation as compared to c-Src indicating the difference in properties between the two members of Src family kinases.
The effect of Hck on p73 induced endogenous gene expression was examined by semi-quantitative reverse transcriptional (RT)-PCR analysis in HeLa cells. Hck and mSH3-Hck were transfected with p73α in a ratio of 6.5:1 and total RNA prepared. These conditions were used to ensure that all p73 expressing cells also co-express Hck. Ectopic expression of p73α resulted in an increase in endogenous Ipaf mRNA levels, and co-expression with WT-Hck resulted in inhibition of expression (Fig. 6F). Co-expression of mSH3-Hck with p73α did not inhibit p73α induced Ipaf mRNA levels (Fig. 6F). This was also observed with endogenous MDM2 mRNA levels where coexpression of WT-Hck inhibited MDM2 gene expression significantly and mSH3-Hck coexpression with p73α did not inhibit p73α induced MDM2 mRNA levels. These results suggested that functional SH3 domain of Hck is indispensable for repression of p73 transcriptional activity. We assessed the ability of Hck to regulate expression of PUMA, a well-known target of p73. RT-PCR analysis showed that p73α expression upregulates expression of PUMA in HeLa cells. Upon co-expression of Hck with p73α, PUMA expression was not altered under the conditions where Ipaf gene expression was repressed (Fig. 6G). This result shows that Hck may regulate endogenous p73 targets selectively.
The effect of Hck on p73 induced Ipaf gene expression in SAOS-2 cells was examined. When Hck was co-expressed with p73α, Ipaf expression was reduced significantly (Fig. 7D). This repression was independent of kinase activity of Hck but dependent on functional SH3 domain of Hck (Fig. 7D). We determined the effect of Y28F-p73α mutation on its ability to transactivate gene expression. Expression of p73α or Y28F-p73α mutant in SAOS2 cells resulted in an increased expression of Ipaf gene as determined by RT-PCR (Fig. 7E). Co-expression of Hck suppressed p73α-induced as well as Y28F mutant-induced expression of Ipaf mRNA (Fig. 7E). To determine whether some other target of p73 is affected by Y28F mutation, we analysed the induction of 14-3-3σ mRNA (known to be induced by p73α)  by p73α as well as by Y28F mutant (Fig. 7E). Induction of 14-3-3σ mRNA was seen upon expression of p73α as well as Y28F mutant. Interestingly, there was no suppression of p73α or Y28Fp73α mutant induced 14-3-3σ mRNA by Hck (Fig. 7E). These results suggest that the suppressive effect of Hck on p73-induced gene expression is promoter specific as was also observed in HeLa cells.
The role of YAP in p73α mediated Ipaf promoter transactivation was determined by knock down of YAP using RNAi strategy. YAP shRNA I, II, III and control shRNA I and II were transfected with GFP-YAP in HeLa cells in the ratio of 4:1 and whole cell lyastes subjected to immunoblotting with GFP antibody. YAP shRNA I, II and III inhibited the expression of GFP-YAP by 90%, 85% and 71%, respectively, as compared to control shRNA I (Fig. 9C). As can be seen in Fig. 9C, (lower panel), transfection efficiency was comparable as determined by GFP blot which was used as an efficiency control. To check the role of endogenous YAP in p73α-induced Ipaf promoter transactivation, YAP shRNA I, II and control shRNA I were transfected with and without p73α along with Ipaf-CAT promoter construct and CAT assays performed after 36 hours of transfection. YAP shRNA I and II significantly inhibited p73α mediated Ipaf-promoter transactivation (P < 0.05) (Fig. 9D). These results suggest that endogenous YAP is required for p73α-induced activation of Ipaf promoter.
The present study identifies p73 as a substrate and interacting partner of the Src family kinase, Hck. In vitro studies using a GST fusion protein suggest that p73α could interact directly with SH3 domain of Hck. Hck represses the transcriptional activity of p73α. This repression was observed under conditions where c-Abl co-expression showed enhanced transactivation by p73 indicating that c-Abl and Hck have opposing influence on the function of p73 as a transcriptional activator. This repression was independent of the catalytic activity of Hck but required its SH3 domain. The effect of Hck on p73 activity was also observed at the level of endogenous Ipaf and MDM2 gene expression but did not influence two other known targets of p73, PUMA and 14-3-3σ. The selective regulation of p73 targets by Hck suggests a distinct role for Hck in vivo with respect to modulation of expression of certain genes. Apoptosis induced by exogenously expressed, as well as endogenous p73, was inhibited by Hck, dependent on its SH3 domain protein interaction function. This property of Hck may therefore be a consequence of its ability to repress target gene induction by p73.
Hck mediated inhibition of p73 activity may be effected by direct interaction between these two proteins in vivo, or by the involvement of a Hck interacting protein that is required for p73 dependent transcriptional activation. Endogenous as well as exogenously expressed Hck has earlier been shown to localize to plasma membrane, Golgi, secretory granules, nongranular membranes and cytosol [47–50]. Most intracellular functions of Src family kinases have been attributed to their localization to the plasma membranes and cytosolic compartments. More recently tyrosine kinases involved in receptor mediated signaling pathways have been shown to be present in the nucleus and their role in influencing activity of transcription factors has been described [54, 61]. Our results suggest that both exogenously expressed as well as endogenous Hck is present to some extent in nuclear compartment of cells. These observations raise the possibility that the inhibition of p73 transcriptional activity by Hck is mediated at least in part by the fraction of Hck present in the nucleus. Cytoplasmic sequestration of p73 by Hck is not likely to explain inhibition of p73 activity by Hck because the level of p73 in the nucleus (which is likely to be the transcriptionally active component) does not decrease upon co-expression of Hck. Hck interaction with p73 directly could alter its ability to bind or transactivate a set of targets selectively.
Our results also show that Hck interacts with YAP, a transcriptional co-activator, which imparts target selectivity to p73. Since YAP was required for maximal activation of Ipaf promoter by p73, it is possible that Hck mediates repression of p73 transcriptional activity through its ability to interact with YAP. Based on the ability of p73δ to interact with Hck and not with YAP , we can infer that Hck and YAP bind to different sequences on p73. Formation of multimolecular complex, whereby both Hck and YAP interact with p73, could be responsible for Hck causing selective repression of p73 target genes. Since Hck can bind to YAP or to p73 through its single SH3 domain, an alternate possibility is that its interaction with YAP may alter the ability of YAP to transactivate p73 targets.
Phosphorylation of p73 by c-Abl at Y99 results in its activation . Our study shows that Hck predominantly targets a tyrosine in the transactivation domain (Y28). This difference in target site beween Hck and c-Abl rules out the possibility of Hck expression resulting in p73 phosphorylation through activation of c-Abl, although Hck is known to activate c-Abl . This modification (Y28 phosphorylation) did not influence the activity of p73 because Y28F mutant showed the same level of activation of Ipaf promoter as WT p73. Hck inhibited Ipaf gene expression induced by p73α as well as Y28F mutant. We also demonstrated that this modification is not a unique property of Hck, since c-Src expression also resulted in predominant phosphorylation of p73 at Y28. In addition, Hck also targets other tyrosine residues (which we have not specifically identified) because Y28 mutant was not totally deficient in phosphorylation.
p73 stabilization has generally been shown to reflect in an increase in its activity [12–14, 24]. Our study showed that over- expression of Hck results in stabilization of p73 protein, dependent on its kinase activity, but this does not lead to an increase in p73 activity. Similarly, it has been reported earlier that interaction of p73 with MDM2 results in p73 stabilization but inhibition of p73 transcriptional activity . Our results also show that activation of endogenous Hck results in tyrosine phosphorylation of p73. p73 protein levels increase upon differentiation of mylemonocytic cells ( and present study). Under these conditions of differentiation, Hck levels and activity are also known to increase. Hck activity mediated signaling may therefore contribute to enhanced protein levels of endogenous p73 upon differentiation. Upon cell fractionation, we observed that Hck co-expression enhanced p73 protein levels only in the cytosolic fraction, but not in the nuclear fraction. Phosphorylated p73 was essentially seen in the cytosolic fraction. p73 localized to nuclear and cytoplasmic compartments may have independent cellular functions just as does p53 [64–66]. Since phosphorylation at Y28 by Hck does not appear to play a significant role in its stability, it is likely that other mechanisms contribute to p73 stabilization dependent on the kinase activity of Hck. Recently, Fyn, a Src family kinase was shown to negatively regulate Itch by tyrosine phosphorylation, impairing its ubiquitinating activity . Since Itch regulates cellular p73 levels by causing its degradation , it is also possible that SFKs may indirectly regulate p73 by preventing its degradation. We have yet to determine the role of p73 stabilized by the kinase activity of Hck in the cytoplasmic compartment.
SFKs modulate diverse signaling cascades dependent on their ability to interact with and phosphorylate various target proteins. In some contexts kinase and adaptor functions can act independently to effect downstream signaling. It therefore, appears that stabilization, which is catalytic activity dependent and suppression of apoptosis, which is SH3 domain interaction dependent, are independent effects of Hck on p73. The functional significance of Y28 phosphorylation of p73 is not clear at present and would require further investigation.
In conclusion, our results show that the tyrosine kinase Hck interacts with p73α physically and functionally. Hck and c-Src phosphorylate p73α at Tyr-28, a novel site of phosphorylation located in the transcriptional activation domain. Transcriptional activity of p73 towards certain target genes is selectively inhibited by Hck independent of its kinase activity. Hck-SH3 domain mediated interactions play an important role in the inhibition of p73-dependent transcriptional activation of target genes as well as apoptosis.
HeLa, Cos1, and human osteosarcoma SAOS-2 cells were grown in Dulbecco's modified eagle medium supplemented with 10% fetal bovine serum and antibiotics. HL-60 cells were maintained in RPMI 1640 medium with heat- inactivated 10% fetal bovine serum and antibiotics. Cultures were maintained in a humidified 37°C incubator with 5% CO2. Transient transfections of HeLa and SAOS-2 were performed with LipofectAMINE Plus reagent according to the manufacturer's recommendations (Invitrogen). For transfection of Cos1 cells cationic lipid DHDEAB was used as described .
The mammalian expression plasmids used were: hemagglutinin (HA) epitope-tagged p73α or p73β in pcDNA3 (gift from Dr. Gerry Melino, University of Rome, Italy), Human p59 Hck in pcDNA6 (gift from Dr. Todd Miller, State University of New York, Stony Brook), c-Src and K297R-c-Src (from Upstate Biotechnology, Lake Placid, New York), GFP-YAP (gift from Dr. Marius Sudol, Mount Sinai School of Medicine, New York), GST-p73α (gift from Dr. Giovanni Blandino, Regina Elena Cancer Institute, Italy), p53/p73 responsive promoter construct PG13-Luc (kindly provided by Dr. Bert Vogelstein, Johns Hopkins University, Baltimore), MDM2-Luc promoter construct (kind gift from Dr. Moshe Oren, Weizmann Institute of Science, Israel), and Ipaf-CAT (pCAT-P2) promoter construct, described by us earlier . Different p73α mutants (tyrosine to phenyl alanine) were made based on tyrosine phosphorylation sites having high scores using the NetPhos 2.0 program for SFK target sites prediction. Y28F-p73α, Y99F-p73α, Y121F-p73α, Y309F-p73α, Y355-56F-p73α, and Hck mutants, K269E-Hck, W93A-Hck were made by PCR based site directed mutagenesis. Plasmids expressing GST-SH3 and GST-mSH3 domain of Hck (amino acids 72-143) were prepared by cloning the appropriate PCR product into BamH1 and EcoR1 sites of pGEX2T. All the sequences of the constructs were confirmed by using an automated DNA sequencer. The green fluorescence protein (GFP) expression plasmid pEGFP-C1 was from Clontech. Antibodies to p73 (H-79, rabbit ployclonal), Hck (rabbit polyclonal), PY20 (mouse monoclonal), α-tubulin (mouse monoclonal), c-Src (goat polyclonal), Calnexin (rabbit polyclonal) and GST (mouse monoclonal) were purchased from SantaCruz Biotechnology. p73 monoclonal antibody clone 1288 was from Imgenex. PARP antibody (rabbit polyclonal) was from Roche.
The YAP shRNA expression vector was constructed using the U6 promoter-based vector essentially as described [56, 71]. The YAP sequence targeted by shRNA (GenBank™ accession NM_006106) was from nucleotides 792-812 (for YPI), 1570-1590 (for YPII) and 1254-1274 (for YPIII). The YAP sequences targeted by shRNAs were a) YPI: 5'-GACATCTTCTCGTCAGAGATA-3' b) YPII: 5'-GCTGCCACCATGCTAGATAAA-3' and c) YPIII: 5'-CCTTAACAGTCGCACCTATCA-3'. The vectors expressing shRNA of unrelated sequence of the same length were used as control. All the sequences were confirmed by automated DNA sequencing.
Cos1 cells transfected with indicated plasmids were lysed in lysis buffer (50 mM Tris-Cl pH 7.5, 150 mM NaCl, 10% glycerol, 0.5%NP-40, 2 mM EDTA, 2 mM EGTA, 2 mM PMSF, 2 mM NaF, 2 mM Na3VO4 and protease inhibitor (Roche Biochemicals) and the extract was incubated with antibodies overnight at 4°C. The immune complexes were captured using Protein A/G Plus beads (Santa Cruz) and washed with buffer (50 mM Tris-Cl pH7.5, 150 mM Nacl, 10% Glycerol, 0.1% NP-40), the proteins were eluted by boiling in 3× SDS-sample buffer and separated on 8% SDS PAGE followed by western blotting with the required antibodies using ECL detection reagent (Perkin Elmer).
For GST pull down assays, cultures of E. Coli DH5α expressing GST, GST-SH3Hck or GST-mSH3-Hck were induced by 1 mM isopropyl-β-D-thiogalactopyranosidase (IPTG) for 4 hours at 37°C. Cells were lysed by addition of cold PBS containing 1 mM PMSF and protease inhibitors (Roche) and sonicated. To this, 1% Triton-X 100 was added for 30 minutes at 4°C for solubilization and then centrifuged to remove insoluble materials. To the supernatant, Glutathione Sepharose beads (50% slurry) were added and incubated with end-to-end shaking on Rototorque at 4°C for 1 hour. Beads were pelleted, washed with PBS containing 0.1% TritonX-100 and incubated 6–8 hours with lysates of Cos1 cells transiently transfected with indicated plasmids. Bound proteins were eluted by boiling in 3× SDS sample buffer and subjected to immunoblotting.
Purified recombinant human Hck (kind gift from Dr. John Kuriyan, UC Berkeley) was activated for 30 minutes at 37°C in 20 μl kinase buffer (10 mM TRIS-Cl pH 7.5, 0.5 mM DTT, 10 mM MgCl2, 1 mM MnCl2) containing 20 μM Na3VO4 and protease inhibitors. Activated Hck (80 nM) was incubated with GST and GST-p73α in the presence of 3 μCi of γ32P-ATP for 30 minutes at 37°C. The reaction was terminated by the addition of 3X-SDS sample buffer followed by boiling for 5 minutes. The proteins were analyzed by 10% SDS-PAGE and gel was stained with commassie blue to visualize expression of proteins and subsequently dried for phosphor image analysis.
HL-60 cells were grown in RPMI medium with 10% heat-inactivated fetal calf serum. These cells were differentiated by the addition of DMSO (1.25%) for 48 hours. Differentiated cells were subjected to HgCl2 treatment as described by Robbins et al. . Briefly, the cells were washed with phosphate-buffered saline and treated with 0.5 mM HgCl2 for 15 minutes at room temperature before the lysates were subjected to immunoprecipitation with p73 (rabbit polyclonal) antibody.
Transfected Cos1 and HL-60 cells were suspended in cold buffer A (10 mM Hepes-KOH pH7.9, 1.5 mM MgCl2, 10 mM KCl, 1 mM DTT, 1 mM PMSF, 2 mM Na3V04, 1 mM EDTA, 1 mM EGTA, protease inhibitors) and incubated on ice for 15 minutes. Later, NP-40 was added to 1% v/v, vortexed for 20 seconds and incubated on ice for 10 minutes. Cells were pelleted down at 1500 g for 5 minutes at 4°C. Supernatant, which is the cytosolic extract, was re-suspended in 3× SDS sample buffer and boiled for 5 minutes. The nuclear pellet was washed thrice with cold buffer A (without NP-40) and re-suspended in 3× SDS sample buffer, boiled for 5 minutes and resolved by 8% SDS PAGE and subsequently immunoblotted with the required antibodies.
Quantitative analysis of apoptotic cells was carried out as described previously [36, 69]. Cells grown on coverslips were transfected and processed for immunostaining using appropriate antibodies for detection of expressing cells. Cells were mounted in 90% glycerol containing 1 mg/ml para-phenylenediamine (antifade) and 0.5 μg/ml DAPI (4'6-diamidino-2 phenylindole) for DNA staining. Cells showing immunofluorescence staining were counted and those cells that showed loss of refraction, condensed chromatin, apoptotic bodies, cell shrinkage were scored as apoptotic. At least 200 expressing cells were counted in each coverslip. The data represent the mean ± S.D. from at least three independent experiments on duplicate coverslips. Background apoptosis was determined by counting non-expressing cells in the same coverslips. For immunofluorescence staining of endogenous Hck protein, HL-60 cells grown on coverslips were fixed after differentiation for 24 hours with 10 ng/ml of 12-O-tetradecanoylphorbol-13-acetate (TPA) and stained with Hck (rabbit polyclonal) primary antibody overnight at 4°C, followed by anti-rabbit Cy3 secondary antibody incubation for 45 minutes.
Hela cells were seeded in 24 well plates and transfected with indicated promoter constructs and expression plasmids along with pCMV.SPORT-βGal (Life technologies, Inc.) Total DNA was kept constant to 400 ng by the use of pcDNA3. Cells were lysed in reporter lysis buffer (Promega, Corp.) after 30 hours of transfection and luciferase activity measured by Luciferase repoter assay system (Promega, Corp). CAT assay was carried out as described previously . Relative luciferase or CAT activities were calculated after normalizing with β-galactosidase enzyme activities.
HeLa and SAOS2 cells were transfected with indicated combination of expression plasmids and after 30 hours of transfection, total RNA was isolated using Trizol reagent (Life Technologies, Inc.). Semi-quantitative PCR was carried out as described previously . Appropriate primers for Ipaf, MDM2, PUMA, 14-3-3σ, Hck, p73 and GAPDH were used for amplification.
We thank, Dr. Gerry Melino, Dr. Giovanni Blandino, Dr. Marius Sudol, Dr. Moshe Oren, Dr. Todd Miller, Dr. John Kuriyan, Dr. Markus Seeliger, Dr. Bert Vogelstein and Dr. Subhashini Sadasivam for providing reagents. PP gratefully acknowledges the Council of Scientific and Industrial Research, India for a senior research fellowship.
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