PHOSPHORYLATION OF VACM-1/CUL5 BY PROTEIN KINASE A REGULATES ITS NEDDYLATION AND ANTIPROLIFERATIVE EFFECT.

Expression of VACM-1/cul-5 gene in endothelial and in cancer cell lines in vitro inhibits cellular proliferation and decreases phosphorylation of mitogen-activated protein kinase (MAPK). Structure-function analysis of VACM-1 protein sequence identified consensus sites specific for phosphorylation by protein kinases PKA and PKC and a Nedd8 protein modification site. Mutations at the PKA specific site in VACM-1/cul-5 ( S730A VACM-1) sequence resulted in increased cellular growth and the appearance of a Nedd8-modified VACM-1/cul5. The aim of this study was to examine if PKA dependent phosphorylation of VACM-1/cul5 controls its neddylation status, phosphorylation by PKC, and ultimately growth. Our results indicate that in vitro transfection of rat endothelial cells, RAMEC, with anti-VACM-1 specific siRNA oligonucleotides decreases endogenous VACM-1 protein concentration and increases cell growth. Western blot analysis of cell lysates immunoprecipitated with an antibody directed against PKA-specific phosphorylation site and probed with anti-VACM-1 specific antibody showed that PKA-dependent phosphorylation of VACM-1 protein

regulated by the posttranslational modifications of their components (11). For example, activation of E3 ubiquitin ligase Itch is regulated by phosphorylation-induced conformational changes (17,18) and COP1 E3 ligase, which affects p53 ubiquitination, is phosphorylated by the ATM kinase (19). The specificity of the ubiquitinproteasome degradation system is further controlled through modification of cullins by Nedd8 protein, which shares 58% identity and 79% similarity with ubiquitin (20)(21)(22). It is now proposed that cullins must be neddylated and form heterodimers to be an active component of the E3 ligase system (14). Conjugation of Nedd8 to cul1 enhances the ability of the complex to promote ubiquitin polymerization and is essential for proteolytic targeting of p27Kip1 (10,(22)(23)(24). Loss of Nedd8 system, on the other hand, leads to the dysfunction of tumor suppression by VHL (25) and compromises cul1 dependent regulation of eye development in Drosophila (26) while in mice it is essential for cell cycle progression (28). In C. elegans development, neddylated cul1 targets katanin, a microtubule severing complex, and thus acts as a negative regulator of contractility and cytokinesis (28,29). Whether modification of cullins by Nedd8 is dependent on their phosphorylation has not been reported.
Analysis of VACM-1/cul5 protein structure revealed a putative modification sequence for Nedd8 at Lys724, protein kinase A (PKA) phosphorylation sequence at Ser730 and Thr426 and fifteen putative protein kinase C (PKC) dependent phosphorylation sites (5). The expression of a VACM-1 mutant where Ser730 has been changed to Ala ( S730A VACM-1) significantly increased cellular growth and created a dominant negative phenotype (5). Further, overexpression of the mutant in rat endothelial cells, RAMEC, converted cells to the angiogenic phenotype when grown on a Matrigel® support (9). These results suggested, therefore, that cellular localization and the biological activity of VACM-1/cul5 protein may depend on its posttranslational modification status by PKA.
The S730A VACM-1 mutant gives us a powerful tool to determine how PKA specific phosphorylation induces modification of VACM-1 by Nedd8 and to elucidate the mechanism of phenotype reversal when expressed in endothelial cells in vitro (5). This approach has been used by others to discover the mechanism by which PKA activity controls localization and activity of proteins that regulate cell growth and angiogenesis (30)(31)(32)(33).
For example, PKA dependent phosphorylation of an oncogene Gli increased its nuclear localization (32), while phosphorylation of a receptor GRK2 recruited the protein to the cell membrane (33). Since E3 ligases determine the specificity of the substrates being targeted for degradation, proteasome inhibitors are now marketed as drugs (15). Consequently, identifying VACM-1/cul5 as a component of the vasculaturespecific E3 ligase, and determining how neddylation and/or phosphorylation affect its localization and biological activity, may be important in identifying specific targets for drugs to control cell growth and angiogenesis.
In this study we report several new findings that will help elucidate the mechanism of VACM-1/cul5 dependent cell growth. First, using siRNA oligonucleotides against VACM-1 mRNA, we confirmed the antiproliferative effects of VACM-1/cul5. Second, we demonstrated that cellular localization of VACM-1/cul5 proteins may be controlled by its posttranslational modifications. Third, we showed that the expression of S730A VACM-1 cDNA, which lacks the PKA specific phosphorylation site, leads to increased neddylation of VACM-1. Finally, we found that PKC induced cell proliferation is significantly higher in cells transfected with S730A VACM-1 cDNA when compared to the control group. Together, these results suggest that preferential phosphorylation of VACM-1 /cul5 protein by these protein kinases regulates its modification by Nedd8 and may allow for the selective regulation of different cellular pathways (5,6,9).
Immunostaining-Affinity purified rabbit polyclonal antibody directed against the Nterminus (Ab-A) of VACM-1 protein (1,8) were used to stain cells by indirect immunofluorescence. Cells grown on coverslips were fixed in 3% paraformaldehyde (in 1xPBS, pH 7.4) for 20 minutes, washed in PBS, permeabilized with 5% Tween-20 solution for 20 minutes, washed with PBS/2% BSA, and incubated for two hours with a 1:20 dilution of Ab-A, or Ab-A preabsorbed with 10 µM peptide A identical in sequence to the amino terminus sequence of VACM-1. Antibodies were diluted in PBS containing 0.1% BSA (PBS/BSA). Cells probed with anti-Nedd8 (Alexis Co.) antibody were treated similarly and exposed to the antibody for 1.5 hrs. The primary antibodies were detected by incubating cells in the presence of 1:40 dilution of either FITC-conjugated goat anti-rabbit IgG or Texas Red conjugated anti-rabbit Ab (Vector Laboratories Inc, Burlingame, CA) in 1X PBS/2% BSA for 1 hr. The slides were washed with 1x PBS with 0.2 % BSA, mounted with Vectashield® mounting medium and viewed by epifluorescence microscopy (Eclipse E600, Nikon) equipped with Spot camera (Diagnostic Instruments, Sterling Heights, MI). The nuclear staining was achieved by DAPI found in the Vectashield® mounting medium (Vector Laboratories Inc, Burlingame, CA). The relative expression of specific proteins was calculated using the NIH Image program (rsb.info.nih.gov/ij/index.html).
Cellular proliferation analysis-RAMEC transfected with VACM-1 cDNA, S730A VACM-1 cDNA, and with CMV vector were seeded in 100 mm plates at equal densities (2x10 4 cells/mL) harvested at specified time points and counted in a hemacytometer. Alternatively, cells were seeded at equal densities in 12-well plates and after three days photographs were taken using Image-Pro Express® at 10x magnification. To quantitate density, cells were counted in at least three random one-square centimeter areas. In addition, cells were grown on coverslips, stained with DAPI as described above, and nuclei visualized under epifluorescence microscopy were counted.
Wound healing growth assay-Cells were plated on 24-well tissue culture plates at 2x10 4 cells/mL per well. After cells reached confluency, the cell layer was scratched using a 1-200 μL pipette tip (38). Cell cultures were photographed at time zero and at sixteen hours after the appropriate treatment, and Image-Pro Express® was used to measure the cell monolayer wound distances. Stock solutions of 1 mM Forskolin (FSK), PMA and Gö6983 were prepared in dimethyl sulfoxide (DMSO) and diluted to appropriate concentrations in tissue culture media immediately before use. The control cells were treated with media containing 0.1% DMSO.
SiRNA-Transfections with anti-VACM-1 siRNAs targeting different regions of the VACM-1/cul5 sequence were performed according to the protocol in the Silencer® siRNA Starter Kit purchased from Ambion® (cat # AM16708A). Three specific antisense oligonucleotides in the Silencer® pre-designed siRNA included: siRNA#1, 5'-AGAUUCCUGGCGUAAAAGCtt-3' (ID 192207), siRNA#2 5'-CCACGUAUCAA GCAUGAGCtt-3' (ID 192208) and siRNA#3 5'-UAGCAUCAUUAACAACUGCtt-3'(ID 192209). The control cells were sham transfected. For the negative control, cells were transfected with siRNAs that did not target any gene sequences provided in the starter kit. The positive control used was GAPDH siRNA provided in the starter kit. In some experiments double transfection was performed after 24 hours and cell lysates were collected at 48 hours (9).
Immunoprecipitation-Total lysates (100-300 µg protein) were prepared from asynchronous cells were resuspended in 150 µL of solubilization buffer (50 mM Tris HCl, pH 8, 150 mM NaCl, 0.3% Triton X-100, 1 mM Pefabloc®SC and 10 µg/ml aprotinin) and incubated with a 1:250 dilution of affinity purified Ab-A (directed against the amino terminus sequence of VACM-1 protein), anti-Nedd8 Ab, anti-phospho-PKA substrate (RRXS/T) Ab (Cellular Signaling Technology) (30) or a nonspecific antibody. After 2 hours of incubation, proteinA/sepharose (Amhersham Pharmacia Biotech) suspension was added and the incubation continued for another 2 hours. The complex was centrifuged at 12,000 RPM for 2 minutes and washed two to three times in the solubilization buffer. Loading dye was added and the immunoprecipitates were heated to 95º C for 5 minutes, separated using 12.5% SDS-PAGE gels, transferred to nitrocellulose and probed with an antibody directed against VACM-1 protein, as described below (5,36).
Western blot analysis-Total cell lysates and membrane fractions were prepared as described previously (5). Cells were grown to at least 70% confluency, washed in ice-cold PBS, and resuspended in 500 μL of buffer (50 mM Tris (pH 7.4), 0.1% Triton X-100, 150 mM NaCl, 1M EDTA, 50 mM NaF) with 1 μg/ml apoprotinin, 100 μ M Pefabloc R SC, and 10 mM PMSF. All samples were homogenized with a Polytron homogenizer and protein concentration was determined using the Bradford method (BioRad Co. Richmond, Ca). Nuclear and membrane fractions were isolated as described previously (35). Both nuclear and membrane pellets were resuspended in the buffer and their protein concentrations were determined as described using Bradford assay (34). All samples were resuspended in 4X sample buffer (Invitrogen® Co.), heated to 95 o C for 5 min, and subjected to SDS polyacrylamide gel electrophoresis (PAGE) using a 12.5% running gel. The separated proteins were transferred to a nitrocellulose membrane (Osmotics Co. Trevose, PA) at 30 mV for 2 hours. Nonspecific binding was blocked by incubating membrane temperature with PBS containing 5% nonfat dry milk and 0.2% Tween-20 for 30 min at room temperature. When probing with antiphospho-PKA phosphorylation specific Ab, blots were blocked with 5% BSA solution. Membranes were next incubated for 2 hours at room temperature in buffer solution containing a 1:200 dilution of affinity purified polyclonal antibodies directed against the N-terminus (Ab-A) of VACM-1 protein (1,36). In some experiments, blots were stripped and reprobed with anti-Nedd8 specific Ab (1:500 dilution) developed in rabbit (Alexis Co.). To ascertain equal protein loading, blots were stripped and incubated in a 1:10,000 dilution of monoclonal anti-mouse GAPDH (Abcam Inc., Cambridge, MA) for 1.5 hours at room temperature. The membranes were next washed in the same buffer for 15 min and twice for 5 min and incubated for 2 hours with a horse radish peroxidase (HRP) conjugated secondary antibodies (diluted at 1:2000 to 1:10,000) (Cell Signaling, Beverly, Ma). The nitrocellulose membranes were washed as described above, exposed to the luminol detection reagents (Cell Signaling, Beverly, Ma) for 1 min or longer, if appropriate, and exposed to the X-ray film (Amhersham, Arlington Heights, Il).
Statistical Analysis-Data are expressed as mean + one standard error (SE) of the mean. SYSTAT® t-tests were used for data analysis. Significance was set at p<0.05 unless noted otherwise.

RESULTS
To confirm the antiproliferative effects of VACM-1/cul5 in vitro, RAMEC expressing endogenous VACM-1 protein (36) were transfected with VACM-1 cDNA. The growth of these cells was monitored as described in Experimental Procedures (9). The CMV vector transfected RAMEC were confluent three days after plating compared to the minimal growth of the VACM-1 cDNA transfected cells (Fig. 1A). When cell growth was quantitated, there was a significant difference in growth rates between the two groups. The VACM-1 cDNA transfected RAMEC failed to reach confluency one week after plating (data not shown).
To further ascertain the antiproliferative effect of VACM-1, we transfected cells with three anti-VACM-1/cul5 specific siRNA oligonucleotides (Ambion® Inc). Time (24 hrs and 48 hrs) and dose (0 nM control, 15 nM and 30 nM) dependent effects of the anti-VACM-1/cul5 specific oligonucleotides were examined using light microscopy and Western blot analysis. Our results (Fig. 2) demonstrate that RAMEC transfected with any of the three anti-VACM-1 specific siRNA oligonucleotides for 24 hours, grew significantly faster than the control cells ( Fig. 2A, n=3 each, *p<0.05). A representative photograph for cells treated with siRNA #2 is shown in Fig. 2B (upper panel). There was no significant difference in RAMEC growth rate between control cells and those cells transfected with the GAPDH siRNA, which was used as a positive siRNA control (Fig.  2B, middle panel). The negative control siRNA, which did not target any particular sequence, was used to evaluate the transfection efficiency, and it had no effect on cell growth ( Fig. 2A, bottom  panel). The growth promoting effects of anti-VACM-1 specific siRNA oligonucleotides were further confirmed at 48 hours after transfection using methylene blue staining technique (Fig. 3A).
To establish that the increase in cell growth was associated with changes in VACM-1 protein concentration, cell lysates from control and anti-VACM-1 specific siRNA oligonucleotide transfected cells were examined for VACM-1 protein expression using the Western blot approach. VACM-1/cul5 signal was quantified and corrected for GAPDH protein concentration. Our results ( Fig. 3B and 3C) show a decrease in VACM-1 protein concentration in RAMEC treated with all oligonucleotides at 24 hours post transfection, but the decrease was statistically significant only in the group treated with siRNA#2 (n=4, p<0.05). The decrease in VACM-1/cul5 protein expression after treatment with siRNA oligonucleotides hours was time dependent. A representative blot for lysates prepared from cells transfected with siRNA#2 and collected at different time points is shown in Fig. 3D. The highest decrease in VACM-1/cul5 protein concentration was observed in RAMEC retransfected with the siRNA at 24 hours and harvested at 48 hours (48-db). To further confirm the effectiveness of the siRNA constructs, we performed immunocytochemistry experiments. The data shown in Fig. 3E further support Western blot analyses results. In cells transfected with all three anti-VACM-1/cul5 siRNAs and stained with anti-VACM-1/cul5 specific Ab at 24 hours after transfection, a clear decrease in the signal was observed with all oligonucleotides tested when compared to the control group. Together, these findings establish the inhibitory effect VACM-1 on cellular proliferation previously described (5,6,9).
Previous work suggested that endogenous VACM-1 is localized to the cell membrane or cytosol when cells are not dividing, and VACM-1 is nuclear when cells are dividing (9,36). To further examine the subcellular localization of VACM-1 protein in RAMEC at different stages of cell cycle, we used a wound healing assay. In this technique, proliferating and nonproliferating cells from the same culture are represented on the same slide. Immunocytochemistry data using anti-VACM-1 Ab suggest that in the proliferating area, VACM-1 protein was localized to the nucleus ( Fig. 4A and Bi), whereas the non proliferating regions VACM-1/cul5 was cytosolic ( Fig. 4A and Bii) or cell-membrane specific (Fig. 4A and Biii). Interestingly, when lysates from asynchronous RAMEC were separated into the nuclear, cytosolic and membrane fractions and separated by SDS-PAGE for immunoblotting with anti-VACM-1 Ab (35), the nuclear but not the cytosolic or the membrane fractions demonstrated presence of a larger M r species that could be recognized by anti-VACM-1 Ab (Fig. 4C). When the signal was quantitated and expressed as a ratio of the upper to lower band, there was a significant difference between the nuclear and membrane or cytosolic fractions (Fig. 4D, n=3, *p<0.05).
The appearance of a larger M r species on Western blot was previously identified as Nedd8 modified VACM-1 protein (5,9). Consequently, wound healing assay described above was used to examine Nedd8 signal in proliferating and nonproliferating cells. Our results indicate that in the wound area where cells are proliferating, Nedd8 signal is very intense and localizes to the nucleus, while in the nonproliferating regions of the cell culture, Nedd8 signal is reduced or absent (Fig.  5A). The coimmunostaining experiments using anti-VACM-1/cul5 specific Ab (green) and anti-Nedd8 specific Ab (red) indicate that VACM-1 and Nedd8 signal colocalize in the nuclear region but not in the cell membrane ( Fig. 5B and 5C).
To investigate this effect further, we next used cell line expressing S730A VACM-1 cDNA mutant (5,9). We have shown previously that in cells transfected with S730A VACM-1 cDNA where PKA specific phosphorylation site has been mutated (5, 9) VACM-1 protein is highly neddylated and localizes to the nucleus. Our immunostaining results shown in Fig. 6A confirm that the Nedd8specific signal was increased in the S730A VACM-1 cDNA transfected RAMEC when compared to the control cells. Further, the immunoprecipitation study where anti-Nedd8 Ab was used to "pulldown" proteins and anti-VACM-1 Ab was used to probe the blot, showed that only the higher M r species was detected (Fig. 6B). These results suggested therefore, that neddylation of VACM-1 protein may be regulated by PKA dependent phosphorylation at Ser730.
Thus, we next explored the hypothesis that the rapid translocation of VACM-1 protein form the nuclear region in non-confluent cells to the cytosol and/or membrane in the confluent cells (Fig. 4) depends on modification of VACM-1 by Nedd8 and is regulated by PKA. To induce PKA activity, CMV and S730A VACM-1 cDNA transfected cells were treated with 10 μM forskolin for 5 or 15 min as described in literature (30,37). Total cell lysates were collected and immunoprecipitated with anti-VACM-1 specific Ab. Samples were resolved on SDS-PAGE and after transfer to nitrocellulose, blots were probed with an antibody that recognizes PKA phosphorylated proteins (30,37). Our results show that PKA dependent phosphorylation of VACM-1/cul5 is higher in the CMV-transfected cells when compared to the S730A VACM-1 cDNA transfected cells (Fig. 6C, first two lanes). PKA specific signal was increased in all experiments from control cells treated with FSK for 5 min. (3787+849 vs 7559+2915, n=4, p<0.2). No change in signal intensity was observed in S730A VACM-1 cDNA transfected cells (2111+703 vs 2024+844, n=3, NS). Interestingly, only the 91 kDa VACM-1/cul5 and not the higher M r species identified in Fig. 6B as the neddylated VACM-1/cul5, was immunoprecipitated with the anti-phospho-PKA substrate specific antibody ( Fig. 6C and D). To extend these experiments even further, we used the immunocytochemistry approach. The representative data shown in Fig.  7A indicate that treatment of control cells with forskolin (FSK, 10 μM) to increase PKA activity, reduced nuclear localization of VACM-1/cul5 protein. This effect was rapid, as VACM-1/cul5 signal intensity was decreased in 45 min and totally disappeared at 2 and 4 hours after treatment. Similarly, we observed that in the proliferating cells (wound area) treated with forskolin for 45 min and stained with anti-Nedd8 specific Ab, Nedd8 signal was reduced as well (Fig. 7B).
To ascertain the specificity of this effect, these experiments were repeated using double immunostaining approach. Our results shown in Fig. 8A indicated that in control cells Nedd8 and VACM-1/cul5 signal in the proliferating cells is largely overlapping. Induction of PKA activity with FSK resulted in a decreased signal intensity. Interestingly, in cells expressing the mutated VACM-1 cDNA lacking the PKA phosphorylation site, treatment with Fsk for 45 min did not appear to affect localization of either protein (Fig. 8B,  lower panel).
In addition to the PKA specific phosphorylation site at Ser730 and the neddylation site at Lys724, 15 putative PKC-specific phosphorylation sites can be identified in VACM-1 sequence (5). To further examine VACM-1 phosphorylation status in S730A VACM-1 cDNA transfected RAMEC, we examined whether VACM-1 signal intensity was affected by treating cell lysates with either 300 μM okadaic acid (OKA) to inhibit endogenous phosphatases, or by treatment with 100U/mL calf-intestinal phosphatase (CIP) to decrease protein phosphorylation (38). Lysate samples pretreated with CIP had lower signal intensity compared to the control or to the OKA treated lysates (Fig.  9A), suggesting that in the absence of PKAphosphorylation site, VACM-1 may still be phosphorylated.
To examine whether PKC plays a role in the regulation of VACM-1 dependent cell growth, cells were treated with PMA to induce PKC activity and with Gö6983 to inhibit its activity (37). The time-dependent and dose-dependant effect of PMA and Gö6983 were established (data not shown). Representative light microscopy results of the wound-healing assay for the CMV control and S730A VACM-1 cDNA transfected cells treated with 10 and 100 nM PMA and 7 nM Gö6983 are shown in Fig. 9B. When data shown in Fig. 9B were quantitated, we observed a significant increase in cell growth in both groups treated with PMA. However the increase in S730A VACM-1 cDNA transfected RAMEC was significantly higher when compared to the control group (n=5, *p<0.05).
Further, when PKC activity was inhibited with 10 nM Gö6983, cell growth was decreased in S730A VACM-1 cDNA transfected cells significantly more than in the control group (n=5, *p<0.05).

Discussion
In this study we report several new findings that enhance our understanding of the mechanism by which VACM-1/cul5 protein regulates cellular by guest on July 8, 2020 http://www.jbc.org/ Downloaded from growth. First, we confirmed that overexpression of VACM-1/cul5 cDNA in rat endothelial cell line inhibits cellular growth, whereas treatment of RAMEC with siRNA oligonucleotides targeting endogenous VACM-1/cul5 leads to an increased cell growth (Figs. 1-3). Second, we demonstrated that the subcellular localization of VACM-1/cul5 protein may be controlled by its posttranslational modifications (Fig. 4). Further, we showed that the nuclear but not cytosol or membrane-localized VACM-1/cul5 protein is modified by Nedd8 protein (Fig. 5). Third, we showed that neddylation of VACM-1/cul5 protein is controlled by PKA specific phosphorylation at S730 (Figs. [6][7][8]. Finally, we found that stimulation of PKC activity with PMA induced cell proliferation significantly higher in cells transfected with S730A VACM-1 cDNA, when compared to the control where PKA-specific phosphorylation site was intact (Fig. 9). Our results suggests for the first time that neddylation of VACM-1/cul5, itself controlled by the PKA dependent phosphorylation, may regulate its subsequent phosphorylation by PKC.
Phosphorylation is a rapid and effective way to change cellular localization and the function of a protein (39). Both PKA and PKC specific phosphorylation regulates cellular function of many proteins in health and disease (30)(31)(32)(33)40). For example, PKA regulates nucleo-cytoplasmic shuttling of a transcription factor Id1 during angiogenesis (32) and is identified as an inhibitory component in the Gli protein translocation to the nucleus (33). Activation of PKA leads to the retention of Gli in the cytoplasm, while inhibition of PKA activity promotes its nuclear localization. Similarly, PKC dependent phosphorylation of specific proteins and its role in cellular processes like angiogenesis, a crucial step in tumor development, has been established (41). Further, the transition from the phosphorylated to the nonphosphorylated form of the Fas-associated death domain-containing protein, associated with carcinogenesis (42), has been used as a marker for cancer progression (43). Although the phosphorylation of proteasome's subunits by specific kinases has been reported (12,16), our results are the first to show that posttranslational modifications of VACM-1/cul5protein by Nedd8 is controlled by PKA-specific phosphorylation.
VACM-1/Cul5 is the least conserved member of the cullin family and its biological significance is only emerging. To date, cul 5 dependent E3 ubiquitin ligase complexes have been shown to control the adenovirus-induced p53 degradation in vitro and the degradation of proteins essential for the prevention of HIV infectivity (44,45). In vivo, expression of VACM-1/cul5 protein is largely endothelium-specific (8,9). When expressed in several cell lines in vitro, VACM-1/cul5 inhibits MAPK phosphorylation and nuclear localization of egr-1, signaling molecules recognized for their role in the regulation of cellular proliferation (5,9). These effects appear to be dependent on the state of VACM-1 modification by Nedd8 as in cells transfected with the S730A VACM-1 cDNA, phosphorylation of MAPK was directly correlated to the level of neddylated VACM-1 protein (5). Interestingly, Nedd8 conjugation to cullins is believed to be fundamental for the activity and stability of numerous E3 ligases (46). Further, it has been reported that cullins must be neddylated and form heterodimers to be an active component of the E3 ligase complex (14). This proposed model supports the dominant negative phenotype observed in S730A VACM-1 cDNA transfected endothelial cells in vitro (5). Importantly, a recent report targeting NEDD8-specific pathway for development of anti-cancer drugs (47), further underscores the significance of our findings.
In summary, this study shows that PKA dependent phosphorylation of VACM-1/cul5 regulates its neddylation and subsequent phosphorylation by PKC. Since the neddylation process is now a target for development of new drugs to regulate excessive cellular proliferation (47), understanding the underlying mechanism at the cellular level is critical.      grown to confluency on glass cover slips were "wounded" with a pipette tip and allowed to grow into the "wound." Immunostaining experiments described in Fig. 4A were repeated using anti-Nedd8 specific Ab.
The immunostaining in the wound and in a confluent region distant from the wound is shown. Magnif., 40X.
B. Cells were fixed and immunostained with anti-VACM-1 specific Ab (green) and anti-Nedd8 specific antibody (red) as described in the Experimental Procedures. Nuclear staining was performed using DAPI (Magnif., 100x). B. Overlay of VACM-1/cul5 and Ned8 proteins localization in the nuclear but not membrane region in control RAMEC.       C.

Nedd8 localization Wound Area Confluent
A.