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Cell Growth & Differentiation Vol. 12, 29-37, January 2001
© 2001 American Association for Cancer Research


Articles

Polo-like Kinase Interacts with Proteasomes and Regulates Their Activity1

Yang Feng, Dan L. Longo and Douglas K. Ferris2

Biological Mechanisms Section, Laboratory of Leukocyte Biology, National Cancer Institute—Frederick Cancer Research and Development Center, Frederick, Maryland 21702 [Y. F., D. K. F.]; National Institute on Aging, Baltimore, Maryland 21224 [D. L. L.]; and IRSP, Scientific Applications International Corp.—Frederick, National Cancer Institute—Frederick Cancer Research and Development Center, Frederick, Maryland 21702 [D. K. F.]

Abstract

The polo-like kinase (Plk) has been shown to be associated with the anaphase-promoting complex at the transition from metaphase to anaphase and to regulate ubiquitination, the process that targets proteins for degradation by proteasomes. In this study, we have identified proteasomal proteins interacting with Plk by mass spectrometry and found that Plk and 20S proteasome subunits could be reversibly immunoprecipitated from both human CA46 cells and HEK 293 cells transfected with HA-Plk. Furthermore, both coprecipitated Plk and baculovirus-expressed Plk were able to phosphorylate proteasome subunits, and metabolic labeling studies indicate that Plk is partially responsible for the phosphorylation of 20S proteasome subunits C9 and C8 in vivo. In addition, phosphorylation of proteasomes by Plk enhanced proteolytic activity toward an artificial substrate Suc-L-L-V-Y-AMC in vitro and in vivo. Finally, we were also able to detect Plk associated with 26S proteasomes under certain conditions. Together our results suggest that Plk is an important mitotic regulator of proteasome activity.

Introduction

The initiation and completion of mitosis is strictly regulated by the cell cycle regulatory machinery, mainly by the cyclin-dependent kinases (1) . In addition to cyclin-dependent kinases, another serine/threonine kinase family, the Plks,3 also play important roles at multiple stages of the cell cycle (2) . After the Drosophila polo kinase was first identified (3) , Plks have been identified in various organisms from yeast to humans (reviewed Ref. 4 ). The protein levels of Plk, its phosphorylation status, and kinase activity are tightly regulated during cell cycle progression (5, 6, 7) .

Early studies found that Plk activity is required for the establishment of a normal bipolar spindle in Drosophila and Schizosaccharomyces pombe (3 , 8) . Microinjection of Plk-specific antibodies into HeLa cells suppresses centrosome separation and decreases levels of {gamma}-tubulin (9) . Cellular localization data also support this function for Plk. Plk is mainly localized at the centrosome in interphase cells, whereas during mitosis, a fraction of Plk is concentrated at the spindle poles and midzone (10) . Other functions of Plks have been reported. Xenopus Plx1 is able to phosphorylate and activate CDC25c, an activator of cdc2/cyclin B complex (7 , 11) . Finally, Plk activity is required for adaptation to DNA damage in yeast (12) and the completion of meiosis and cytokinesis (13, 14, 15) .

Recent studies have emphasized that Plk functions at late stages of the cell cycle, especially in regulating the APC or ubiquitin ligase system. It has been reported that cdc2/cyclin B-activated Plk is able to phosphorylate three components of the APC and improve the ability of the APC to ubiquitinate cyclin B in vitro (16 , 17) . In Saccharomyces cerevisiae, cdc5 mutant cells display a defect in the destruction of mitotic cyclins and a reduction in the cyclin-ubiquitin ligase activity of the APC (18) . In Xenopus egg extracts, Plx1 is required for the APC-mediated degradation of cyclin B2 and cut2, both of which are important for the M-to-I phase transition (19) . Therefore, in part, Plk regulates the completion of mitosis via its regulation of ubiquitination of target proteins, a step preceding proteolysis by proteasomes.

The 26S proteasome is composed of a 20S core catalytic complex and 19S regulatory caps. The 20S proteasome is a Mr 700,000, barrel-shaped structure, formed by two {alpha}-rings on the outer side and two ß-rings on the inner side of the barrel (20) . Each ring is composed of seven small proteins with molecular weights of Mr 21,000–32,000. Together with the 19S regulator, proteasomes are responsible for proteolysis of most ubiquitinated proteins. Substrates of proteasomes include cell cycle regulatory proteins, antigens, and misfolded or damaged proteins. Thus, their activity is important for cell cycle progression, metabolism, and immune responses. However, the mechanisms regulating the activity of proteasomes are not well understood. Phosphorylation of proteasome components C8 and C9 seems to increase proteolytic activity, but the kinase responsible for the phosphorylation has not been identified (21 , 22) . Casein kinase II was found to copurify with 20S proteasomes and to phosphorylate a Mr 30,000 proteasome subunit; however, whether the phosphorylation mediated by casein kinase II affects proteasome activity is still in question (23) . Further work is needed to understand the correlation of proteasome subunit phosphorylation with proteolytic activity. Other regulatory mechanisms may include subunit composition switches induced by IFN-{gamma}, differential association of 20S with 19S or 11S regulatory complexes, and changes in steady-state levels of individual proteasome proteins (24) .

In a previous report, we showed that Plk can be detected in a multiprotein complex, and we identified {alpha}-, ß-, and {gamma}-tubulins as partners in the complex (25) . Other groups have reported several additional proteins interacting with Plk at M phase, including mitotic kinesin-like protein-1 (6) , members of the APC (17) , peptidyl-prolyl isomerase 1 (26 , 27) , and a microtubule-associated protein (28) . It is likely that Plk is a member of several distinct macromolecular complexes that regulate discrete functions. Identification of such complexes should be helpful in understanding the unknown aspects of Plk functions. In this report, we show that Plk is stably associated with 20S proteasomes and that Plk-mediated in vitro phosphorylation of proteasome subunits increases proteolytic activity and that such regulation also occurs in vivo.

Results

Identification of Proteasome Components as Plk-associated Proteins in Human CA46 Cells.
Plk polyclonal antibody (Zymed) was raised against a COOH-terminal Plk peptide, it recognized a Mr 64,000 protein in CA46 cell lysates (for the antibody specificity, also refer to Ref. 6 ), and preincubation of the antibody solution with the cognate COOH-terminal peptide abolished the signal on Western blot (Fig. 1Citation ,Lanes 1 and 2). This Plk antibody was used to immunoprecipitate Plk from mitotic cell lysates (Fig. 1Citation , Lanes 3 and 4). In addition to Plk, several other proteins were consistently coimmunoprecipitated. More importantly, these proteins were specifically competed when Plk COOH-terminal peptide was included in the immunoprecipitation. Our focus was placed on several low molecular weight proteins at the Mr 30,000–36,000 range (bands 1 to 3) that were clearly competed by the Plk peptide. These proteins were excised from the gel, reduced and alkylated with iodoacetic acid, digested with sequencing grade trypsin, and analyzed by mass spectrometry. Sequences obtained matched the following 20S proteasome subunits in the NCBI protein database: xapc7, {zeta}, {iota}, and C3 of {alpha}-subunits; C5 and C10 of ß-subunits (Table 1)Citation . Among the six subunits identified, four subunits had 13% or more of their total amino acid sequences positively identified. Several nomenclatures have been used to name proteasome subunits; we will adopt the system used by Kopp et al. (20) , Affiniti (where most of proteasome subunit antibodies used in this study were obtained), and others (29, 30, 31) . According to this nomenclature, the seven {alpha}-subunits of the human 20S proteasome are: C2, C3, C8, C9, xapc7, {zeta}, and {iota}. We detected all but C3 in Plk immunoprecipitates by immunoblotting (Fig. 2A)Citation . The C3 antibody only detected a very weak signal in total lysates (data not shown), and we expect that C3 is present in this complex. The seven ß-subunits are C5, C7, C10, N3, X, Y, and Z. Among them, subunits X, Y, and Z could individually switch to LMP7, LMP2 (or delta), and MECL-1 when induced by IFN-{gamma} (24) . Subunits C10, N3, X, Y, and Z were detected in Plk immunoprecipitates by immunoblotting. No antibodies specifically recognizing subunits C5 and C7 are currently available. Therefore, 13 of 14 types of subunits were positively identified to be associated with Plk either by sequence data or by immunoprecipitation/immunoblotting. All 11 subunits detected by coprecipitation were competed, along with Plk, by including excess COOH-terminal peptide in the immunoprecipitation. Similar results were observed in S-phase and prometaphase cells, although the amounts of Plk were reduced in interphase cells (data not shown).



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Fig. 1. Plk is associated with multiple low molecular weight proteins. Mitotic CA46 cell lysates separated on a 10% SDS-PAGE were probed with Plk antibody (Lane 1) or a Plk antibody solution preincubated with cognate COOH-terminal peptide (Lane 2). Mitotic lysates were subjected to immunoprecipitation with the Plk antibody in the absence (Lane 3) or presence (Lane 4) of excess cognate peptide. Lane 5 was a mock immunoprecipitation with nonspecific IgG. The immunoprecipitates were resolved by 10% SDS-PAGE, and the gel was stained with Coomassie brilliant blue R-250. Arrows, proteins coprecipitated with Plk but competed by the peptide. Proteins 1, 2, and 3 were collected for mass spectrometry analysis. *, Mr 200,000 nonspecific protein associated with the antibodies. In Lane 3, IgG heavy chain comigrated with coprecipitated tubulins. Molecular weight standards are indicated between lanes in thousands.

 

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Table 1 Subunits of the 20S proteasome identified by mass spectrometry analysis

 


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Fig. 2. Reversible coimmunoprecipitation of proteasome subunits and Plk from CA46 cell lysates. A, Plk immunoprecipitates from CA46 cell lysates were resolved by 10% SDS-PAGE and probed with antibodies specific to individual {alpha}- or ß-subunits of the 20S proteasome. Antibodies to the Y and X subunits were polyclonal rabbit antibodies; all others were mAbs. In the last lane, normal IgG was used as immuoprecipitation (IP) antibody control and probed with Ab to C2. B, CA46 cell lysates were subjected to immunoprecipitation (IP) with antibodies to proteasome subunits {zeta}, C8, C9, or nonspecific IgG (C), and the precipitates were probed with Plk antiserum. C, CA 46 cells were lysed in a low salt buffer containing ATP and immunoprecipitated with Plk antibody or mAb to the {zeta} subunit of the proteasome. The immunoprecipitates were analyzed by Western blot with pAb to subunit 7 of the 19S regulator.

 
To eliminate the possibility that coprecipitated proteasomes were detected because of their nonspecific binding to the Plk antibody used in the immunoprecipitation, reverse immunoprecipitations were carried out with the proteasome antibodies. We tested several proteasome subunit antibodies capable of immunoprecipitating the 20S proteasome. Antibodies specific to 20S proteasome subunits {zeta}, C8, and C9 coprecipitated small amounts of Plk, which were only visualized after prolonged exposure of the Western blot (Fig. 2B)Citation . This weak detection of Plk in proteasome precipitates may be attributable to the abundance of proteasome protein (up to 1% of the cell protein content; Ref. 29 ), compared with low levels of Plk in cell lysates (<0.03% of the total cell mass). Alternatively, Plk could be associated with only a specific subset of proteasomes.

Proteasomes have two main isoforms in cells: 20S and 26S (20S-19S). We tested for the presence of the 19S regulator in the Plk immunoprecipitates. No 19S regulator was detected in Plk immunoprecipitations performed in NP40 lysis buffer. Because the 26S proteasome requires ATP and low salt for its stability (32 , 33) , cells were homogenized in a low salt buffer [20 mM Tris/HCl (pH 7.0), 5 mM ATP, 1 mM ß-mercaptoethanol, 0.1 mM EDTA, and 20% glycerol], and a similar immunoprecipitation was repeated in this buffer. Under these conditions, ATPase subunit 7 of the 19S regulator was indeed detected by immunoblotting 20S-{zeta} and Plk immuoprecipitates (Fig. 2C)Citation . Therefore, it appears that given the conditions under which 26S proteasomes remain intact, Plk is associated with the 26S particles containing the 19S regulatory caps. In this study, we focus our attention on the Plk/20S proteasome interaction because the 20s proteasomes carry the core catalytic activity, and most 19S subunit antibodies from Affiniti only recognize yeast proteins.

Association of HA-tagged Plk with Proteasomes in Transfected HEK 293 Cells.
To further investigate the specificity of Plk/proteasome association, we transfected HEK 293 cells with HA-tagged wild-type Plk, kinase-inactive mutant K82R, or kinase domain-deleted fragment. All these constructs were highly expressed in 293 cells (Fig. 3A)Citation . Both wild-type and K82R were coimmunoprecipitated with the proteasome, whereas deletion of the kinase domain nearly abolished the interaction (Fig. 3B)Citation . Similarly, HA-Plk and HA-K82R were also coprecipitated by an antibody to the proteasome "core." In the reciprocal experiment, proteasomes were coprecipitated with HA-Plk and HA-K82R by the HA tag antibody (Fig. 3C)Citation . Again, in cells transfected with full-length Plk and K82R, coprecipitated proteasome subunits were easy to detect, whereas it required much longer exposure of the Western blot to detect subunits coprecipitated with {Delta}KD (data not shown). Therefore, with increased expression of Plk protein in transfected HEK 293 cells, proteasomes, and full-length Plk can be reversibly and efficiently coprecipitated. These results indicate that the presence of proteasomes in Plk immunoprecipitates observed in Figs. 1Citation and 2Citation was not attributable to cross-reaction with the Plk antibody. The reduction in coprecipitation seen with the Plk kinase domain deletion mutant indicates that the interaction of Plk and proteasomes requires the kinase domain, but not its activity, in binding to the large 20S structure.



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Fig. 3. Immunoprecipitation and immunoblotting of HA-Plk and 20S proteasomes in HEK 293 cells. HEK 293 cells were transfected with vector control (V), HA-tagged, full-length Plk (Plk), inactive kinase mutant (K82R), or kinase domain (amino acids 53–305) deleted fragment ({Delta}KD). A, Western blot of total lysate with HA tag mAb. B, equal amounts of total lysate proteins were used for immunoprecipitation with mAb specific to subunit {zeta} or the "core" of the 20S proteasomes. Gel-resolved samples were then blotted with HA tag antibody to detected coprecipitated Plk. C, lysates were subjected to immunoprecipitation with HA tag antibody, and the immunoprecipitates were probed with mAb specific to subunit {zeta} of the 20S proteasomes. Left, molecular weight standards (in thousands). Arrowheads, Plk proteins. H, IgG heavy chain; L, IgG light chain.

 
The Proteasome Is Phosphorylated by Coprecipitated Plk and Recombinant His6-Plk in Vitro.
Having demonstrated a specific association between Plk and the 20S proteasome, we next tested the ability of endogenous Plk to phosphorylate coprecipitated proteasomes. When Plk immunoprecipitates were subjected to kinase reactions with [{gamma}-32P]ATP, autoradiography revealed phosphorylated proteins of Mr ~32,000 (data not shown). To identify the phosphorylated proteins, the kinase reaction mixture was boiled in 1% SDS to disrupt protein associations, and antibodies specific to individual proteasome subunits were added to the mixture for reimmunoprecipitation. Previous reports have shown that {alpha}-subunits C8 and C9 of the 20S proteasome are phosphorylated in vivo (22) . Reimmunoprecipitation with monoclonal antibodies specific for C8 and C9 isolated phosphorylated proteins of the expected sizes (Fig. 4A)Citation , whereas no phosphorylated {zeta} or C2 were detected. The polyclonal antibody to the 20S proteasome "core" recognizes an unnamed proteasome protein of Mr 30,000. This "core" antibody reprecipitated a phosphorylated Mr 30,000 protein.



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Fig. 4. Phosphorylation of proteasome subunits C8 and C9 by coprecipitated Plk. A, Plk immunoprecipitates from mitotic CA46 cells were subjected to kinase reactions. The reaction mixtures were boiled in SDS and further immunoprecipitated with proteasome subunit antibodies specific to C8, C9, {zeta} C2 (C2), core, or a nonspecific IgG control (control). Autoradiography revealed the phosphorylated band (arrow) recognized by antibodies to C8, C9, and "core." B, HEK 293 cells were transfected with vector control (V), HA-tagged inactive kinase mutant (K82R), or full-length HA-Plk (Plk). Lysates were made and immunoprecipitated with HA antibody and subjected to in vitro kinase reaction. Autoradiography is shown. C, Coomassie staining of HA immunoprecipitates from 293 cells.

 
To test whether other kinases potentially present in the immunoprecipitates might be responsible for phosphorylating proteasome subunits, we transfected HEK 293 cells with HA-tagged Plk and K82R (kinase-inactive mutant with a single amino acid change). Kinase reactions performed after immunoprecipitation of these recombinant proteins indicated that proteasome subunits were only phosphorylated in immunoprecipitates containing functional Plk kinase (Fig. 4B)Citation . Profiles of Coomassie-stained proteins associated with HA-Plk and HA-K82R were essentially identical (Fig. 4C)Citation . These results suggest that Plk is directly responsible for phosphorylation of proteasome components in vitro.

We next tested the ability of purified recombinant Plk to bind to and phosphorylate proteasomes. His6-Plk was purified from insect cells with nickel-chelating beads and incubated with CA46 mitotic lysates at 4°C or 37°C, and beads were washed extensively after incubation. Proteins bound on beads were released and analyzed by immunoblotting. The C8 subunit was detected on beads incubated at 37°C (Fig. 5Citation A, Lane 2), whereas it was not present in beads incubated at 4°C (Fig. 5Citation A, Lane 1). Probing with other proteasome subunit antibodies produced similar results (data not shown). Lanes 3 and 4 show that a similar amount of His6-Plk was incubated with cell lysates at 4°C or 37°C. Thus, recombinant Plk can associate with proteasomes in vitro but only at physiological temperature.



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Fig. 5. Recombinant His6-Plk activated by okadaic acid treatment and M-phase lysate phosphorylate proteasome subunits. A, recombinant His6-Plk on ProBond beads was incubated with M-phase CA46 lysate at 4°C (Lane 1) or 37°C (Lane 2). Bound proteins were released from the beads with SDS sample buffer and resolved by 10% SDS-PAGE. Immunoblot with antibody to proteasome subunit C8 is shown. Coomassie staining of His6-Plk was used for incubation at 4°C (Lane 3) or 37°C (Lane 4). B, Lanes 1–5, autoradiography of kinase reactions performed with recombinant His6-Plk. Lane 1, His6-Plk + purified 20S proteasomes; Lane 2, His6-Plk from insect cells treated with okadaic acid + purified 20S proteasomes; Lane 3, His6-Plk incubated with M-phase lysate at 4°C as in Lane 1 in A; Lane 4, His6-Plk incubated with M-phase lysate at 37°C as in Lane 2 in A; Lane 5, His6-Plk incubated at 37°C with M-phase lysate immuno-depleted with proteasome proteins; Lane 6, Coomassie staining of purified 20S proteasome used in Lanes 1 and 2. C, Coomassie staining of His6-Plk preparation without (Lane 1) and with okadaic acid treatment (Lane 2). Left in all panels, molecular sizes in thousands.

 
The recombinant His6-Plk had autophosphorylation ability and efficiently phosphorylated {alpha}-casein (10) , but it did not phosphorylate purified proteasome preparations (Fig. 5Citation B, Lane 1). However, His6-Plk activated by okadaic acid treatment of insect cells had higher kinase activity toward {alpha}casein and reduced mobility on SDS-PAGE (data not shown), similar to that observed for Plk immunoprecipitated from mitotic cells (16) , and such activated Plk was able to phosphorylate exogenously added proteasomes (Fig. 5Citation B, Lane 2). We preincubated His6-Plk purified from nontreated insect cells with CA46 M-phase lysates to take advantage of the fact that proteasomes bind to His6-Plk beads at 37°C to see whether His6-Plk could be activated by endogenous CA46 lysate factors. It was found that when the preincubation took place at 37°C, His6-Plk subsequently phosphorylated proteasome proteins captured from the cell lysate (Fig. 5Citation B, Lane 4). Six phosphorylated protein bands between Mr 28,000 and Mr 36,000 were detected in Lane 4, a pattern typical of purified 20S proteasome protein profiles on Coomassie-stained gels (Fig. 5Citation B, Lane 6). Immunodepletion of proteasomes from the M-phase lysates before the preincubation removed the six phosphorylated proteins (Fig. 5Citation B, Lane 5). Lanes 1 and 2 in Fig. 5CCitation show the purity of His6-Plk prepared from untreated or okadaic acid-treated insect cells. These data indicate that activated His6-Plk is able to associate with endogenous proteasomes from human CA46 cell lysates and to phosphorylate proteasome subunits. Because cdc2/cyclin B has been reported to phosphorylate and activate Plk to phosphorylate APC (17) , we also treated His6-Plk with purified cdc2/cyclin B. Although we found that His6-Plk was phosphorylated by cdc2, we detected no activation of the recombinant Plk in phosphorylation of added purified proteasomes (data not shown).

We have quantified the amount of proteasome subunits C8 and C9 phosphorylated by His6-Plk in vitro by two-dimension immunoblotting. The in vitro kinase reaction mixtures containing okadaic acid-activated His6-Plk and 20S proteasome proteins were separated on NEPHGE and 12% SDS-PAGE. Phosphorylated proteins were detected by autoradiography, and the total C8 and C9 proteins present in the reaction were subsequently revealed with immunoblotting. Autoradiography revealed two phosphorylated proteins at Mr 31,000 (Fig. 6A)Citation . These phosphorylated proteins were later confirmed to be proteasome subunits C8 and C9 by immunoblotting with antibodies specific to C8 and C9 (Fig. 6, B and C)Citation . Both C8 and C9 have multiple forms migrating at pH ranges of 4.5 to 7.5, and 4.5 to 5.9, on this NEPHGE system. We found that the vast majority of C8 was already phosphorylated when the 20S proteasomes were purified (Fig. 6D)Citation , whereas phosphorylated C9 was not detected in the purified preparation (Fig. 6E)Citation . Phosphatase treatment of the proteasome proteins leads to the accumulation of basic forms (data not shown). The immunoblots in Fig. 6, B and CCitation , were scanned by a densitometer, and the areas matching the phosphorylated proteins were calculated. About 16% of subunit C9 was phosphorylated by His6-Plk in vitro under the experimental conditions. We estimated that the percentage of C8 phosphorylated by His6-Plk was <5%. This low level of phosphorylation correlates well to the modest change in the proteasome activity induced by Plk-mediated phosphorylation of proteasomes in vitro (see results in Table 2Citation ).



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Fig. 6. Two-dimensional NEPHGE analysis of phosphorylated proteasome subunits C8 and C9. The in vitro kinase reaction containing recombinant His6-Plk and purified 20S proteasome was separated by two-dimensional gel electrophoresis. Gels were transferred to Immobilon-P for autoradiography (A). The same blot was then probed with mAb to C8 (B) and stripped and reprobed with mAb to C9 (C). Arrows 1 and 2, phosphorylated proteasome proteins on the autoradiography and corresponding positions on the immunoblots. The 20S proteasomes used in the test shown in A were analyzed by 2-dimensional immunoblotting with antibodies specific to C8 (D) and C9 (E). The numbers on the top indicate the pH gradient in the NEPHGE gel, and numbers on the left are molecular weight markers (in thousands).

 

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Table 2 Effects of Plk-mediated phosphorylation of proteasome subunits on proteasome activitya

 
Plk Phosphorylates 20S Proteasome Subunits C9 and C8 in Vivo.
We have shown that immunoprecipitated endogenous Plk and recombinant Plk are able to bind to and phosphorylate proteasome subunits in vitro. To examine whether this Plk-mediated phosphorylation occurs in cells, we performed 32P metabolic labeling experiments in HEK 293 cells transfected with tagged wild-type HA-Plk or inactive kinase mutant HA-K82R. Autoradiography of proteasomes immunoprecipitated by a mixture of proteasome subunit antibodies showed that proteasomes were heavily phosphorylated in cells transfected with control vector and HA-Plk, whereas they were somewhat less phosphorylated in cells transfected with HA-K82R (Fig. 7A)Citation . A duplicate set of samples was treated with 1% SDS to disrupt protein associations and subjected to reimmunoprecipitation with mAb specific to proteasome subunits C9 and C8. Fig. 7BCitation shows that phosphorylation of subunit C9 was completely abolished in cells transfected with HA-K82R. In contrast, phosphorylation of subunit C8 (Fig. 7C)Citation in cells transfected with HA-K82R was not greatly reduced from control cells, although the wild-type transfections showed a substantial increase over the controls. These results suggest that Plk plays a significant role in phosphorylating proteasomes in vivo.



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Fig. 7. Plk phosphorylates proteasome subunits C9 and C8 in vivo. HEK 293 cells were transfected with control vector pcDNA3 (V), tagged wild-type HA-Plk (Plk), or inactive kinase mutant HA-K82R (K82R). Cells were labeled with [32P]orthophosphate, and lysates were made in NP40 lysis buffer. Proteasomes were immunoprecipitated with a mixture of pAb to "core" and mAb to {zeta} subunit. Autoradiography of immunoprecipitates (A) is shown. After the first immunoprecipitation (IP), beads were boiled in 1% SDS to denature all proteins and diluted in RIPA buffer. The resulting supernatant was subjected to another round of immunoprecipitation with mAb to C9 (B) or mAb to C8 (C), and autoradiography is shown. Left, molecular weight standards (in thousands).

 
Phosphorylation of Proteasome Subunits by Plk Enhances Proteolytic Activity.
Although there have been several reports on the phosphorylation of proteasome subunits, no definitive correlation between this modification catalyzed by specific kinases and proteasome activity has been reported. We purified 20S proteasomes to near homogeneity from asynchronous CA46 cells. On Coomassie-stained gels, the purified 20S proteasome displayed the characteristic 6–7 protein bands between Mr 21,000 and Mr 32,000 (Fig. 5Citation B, Lane 6). Purified proteasome preparations (1 µg/reaction) were subjected to phosphorylation by activated His6-Plk, or mock reactions, followed by degradation assays as described in "Materials and Methods." The fluorogenic artificial substrate Suc-L-L-V-Y-AMC was used to monitor proteasome activity. As seen in Table 2ACitation , phosphorylation by okadaic acid-activated His6-Plk increased the proteolytic activity of 20S proteasomes by 2-fold, and phosphorylation by M-phase-activated His6-Plk increased activity by 1.5-fold. Numbers in Table 2ACitation represent results from five independent experiments.

Having shown that Plk is able to phosphorylate proteasome subunits in vivo and Plk-mediated phosphorylation regulates proteasome activities in vitro, we tested proteasome activities in 293 cells transfected with HA-Plk or HA-K82R (Table 2B)Citation . Proteasomes were immunoprecipitated with the polyclonal antibody to the "core" and monoclonal antibody to the {zeta} subunit of the proteasome in the presence of phosphatase inhibitors in the lysis buffer. Similar experiments were repeated five times. Proteolytic activities associated with immunoprecipitated proteins are shown. Cells transfected with HA-Plk displayed higher proteolytic activities than vector control cells, whereas cells transfected with HA-K82R had lower (20% reduction) activity than the control cells, suggesting that Plk-mediated phosphorylation of proteasomes regulates their activity in vivo. These modest alterations in activity probably relate to the fact that not all of the cells in the culture are effectively transfected, and not all proteasomes are associated with recombinant Plk. Interestingly, we found that without the phosphatase inhibitors, okadaic acid and calyculin A, in the lysis buffer, only a minimal amount of proteasome activity was detected, implying that phosphorylation is an important regulatory event for the ubiquitin-proteasome pathway.

Discussion

We have demonstrated that Plk interacts with 20S proteasomes. This is supported by both mass spectrometry analysis of coimmunoprecipitated proteins from CA46 cells and reciprocal immunoprecipitation/immunoblotting in both CA46 and Plk-transfected HEK 293 cells. Overall, our results demonstrate that Plk functionally interacts with the 20S proteasome and positively regulates proteasome activity. This is a novel role for Plk in cell cycle regulation and a new mechanism for coordinating proteasome activity through the cell cycle. Like Plk, proteasomes are present in the nucleus as well as the cytoplasm in different types of cells (34) . Cytoplasmic proteasomes can be found in the endoplasmic reticulum, Golgi apparatus, and intermediate filaments. The distribution of proteasomes undergoes changes during cell cycle progression. In mitotic cells, proteasomes are found associated with the spindle poles and chromosomes (35 , 36) , which are also major localization sites of Plk (10) . Thus, colocalization of proteasomes and Plk is compatible with our findings that Plk interacts with proteasomes. Results from coimmunoprecipitation studies indicate that only a fraction of total proteasomes are associated with Plk; however, this small portion could be localized to critical sites, such as spindle poles and kinetochores.

Previously, mammalian Plk and yeast Cdc5 have been shown to regulate ubiquitination of mitotic cyclins through the phosphorylation of the APC (16 , 17) . Both Plk and Cdc5 are substrates of the ubiquitin pathway (18 , 37 , 38) . In this study, association of Plk with proteasomes was detected during S phase and prometaphase, times when Plk protein is not degraded, indicating that it is not a transient association occurring just before degradation of Plk. Furthermore, the Plk we detected associated with proteasomes is not itself ubiquitinated (Fig. 1)Citation . Therefore, the association we demonstrate in this study between Plk and proteasome is not attributable to proteasome-mediated degradation of Plk.

We have shown that coprecipitated Plk and recombinant Plk were able to phosphorylate subunits of the 20S proteasome and have identified subunits C8 and C9 to be two of the subunits phosphorylated by Plk in vitro and in vivo. We observed ~16% of subunit C9 phosphorylated by recombinant Plk in vitro. This moderate level of phosphorylation correlates well to the 1.5–2-fold change in the proteasome activity induced by Plk-mediated phosphorylation. Although the general change in proteolytic activity is modest, we expect that the small fraction of proteasomes associated with and regulated by Plk in vivo is localized to important mitotic machineries: spindle pole and kinetochores. Thus, the effect of Plk on proteasome activity could be significant. Both Figs. 6Citation and 7Citation indicate that Plk is more important to the phosphorylation of subunit C9 than to that of C8. Therefore, it seems that proteasome subunit C9 is a preferred substrate for Plk. Recombinant Plk phosphorylated multiple proteins of purified proteasomes, compared with a single phospho-band detected that coprecipitated with Plk. This may be attributable to: (a) proteasomes immunoprecipitated from M-phase lysates that are already partially phosphorylated; (b) limited amounts of Plk and proteasomes in coprecipitated samples; and (c) the abundance of purified proteasome proteins in kinase reactions involving recombinant Plk. In the in vitro activity test, we observed an increased proteolytic activity in proteasomes phosphorylated by activated Plk. Thus, it appears that Plk is both a substrate, as shown previously (37 , 38) , and a regulator of proteasomes. It is noteworthy that we only detected 20S proteasomes associated with Plk and not 26S proteasomes containing the 19S regulatory caps, when cells were lysed in the NP40 lysis buffer. This finding is explained by the instability of 26S proteasome particles in the high salt buffer lacking ATP. Our results indicate that Plk does interact with the intact 26S complex under low salt conditions. Because the 20S proteasome harbors the core catalytic activity of proteasomes, the effect of Plk phosphorylation may have profound physiological consequences. Direct control of proteasome activity through Plk-mediated phosphorylation of 20S subunits may be crucial to mitotic progression. Alternatively, phosphorylation of C8 and C9 may help the binding of the outer {alpha}-ring of the 20S proteasome to the 19S regulator and assembly of the 26S proteasome. This speculation is supported by a previous report showing that phosphorylation of C8 and C9 is even higher in the 26S proteasome than in the core catalytic complex (22) . Lastly, it is also possible that phosphorylation of proteasomes might be required for the translocation of proteasomes in cells.

Proteolysis of regulatory proteins at distinct times is crucial to normal cell cycle progression and organism development. An obvious example is the destruction of cyclin B1, which is required for exit from mitosis (1 , 39) . Thus, it is extremely important that the proteasome activity be properly controlled. It has been reported that in dividing cells, a dramatic increase of proteasome proteins associated with spindle microtubles is observed in early and late anaphase (35) . The transition from metaphase to anaphase requires a rapid increase in proteasome activity, which cannot be achieved easily by coordinated synthesis of multiple proteasomal proteins. A posttranslational modification seems to be a physiologically desirable way to rapidly achieve a robust proteolytic activity and relocalization of proteasomes. In this context, Plk appears to be a good candidate to carry out the phosphorylation for multiple reasons: (a) Plk protein degradation occurs later than degradation of cyclin B1 during mitosis (6) ; (b) redistribution of proteasomes from the interphase patterns to multiple sites on the mitotic machinery is similar to Plk redistribution; (c) Plk phosphorylates the same subunits of the 20S proteasome in vitro and in vivo that have been reported previously to be phosphorylated in vivo; and (d) cells transfected with wild-type Plk show increased proteasome phosphorylation and proteolytic activity, whereas cells transfected with mutant inactive Plk show decreased phosphorylation of proteasome components and decreased activity. Indeed, competing endogenous Plk activity with dominant-negative HA-K82R prolongs mitosis of 293 cells (data not shown).

Several Plk-interacting proteins have been reported, including mitotic kinesin-like protein (6) , a microtubule-associated protein (28) , tubulins (25) , components of APC (17) , and Pin1 (26 , 27) . These proteins, together with proteasomes, are likely key players in linking Plk to its functions in regulation of spindle formation and completion of mitosis. At present it is difficult to evaluate the relationships among the Plk-containing complexes that have been described. In the present studies, we found that the Plk-20S proteasome complex did not contain tubulins (data not shown). It is thus likely that Plk is a component of several multimolecular complexes allowing for its versatile functions during cell cycle progression. We are currently attempting to identify several other proteins that are consistently coimmunoprecipitated with Plk and which may represent additional types of cell cycle regulatory complexes.

Materials and Methods

Cell Culture and Lysates.
Human B-cell lymphoma cells, CA46, were maintained in RPMI 1640 supplemented with 10% FBS. Prometaphase cells were obtained by treating cells with nocodazole (100 ng/ml) for 16 h. To synchronize cells at the G1-S transition, cells were blocked with aphidicolin (0.75 µM) for 16 h, released for 8 h, and then blocked with thymidine (2.5 mM) for 16 h. Cells at G1 phase were obtained by synchronization with mimosine (300 µM) for 16 h. Cells were lysed in NP40 lysis buffer [50 mM Tris (pH 7.4), 1% NP40, 250 mM NaCl, 10 mM NaF, 5 mM EDTA, 1 mM DTT, 1 µg/ml leupeptin, 1 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride] on ice for 1 h. Lysates were centrifuged at 100,000 x g for 1 h at 4°C. The clarified supernatant (S100) was used for immunoprecipitation experiments.

Immunoprecipitation.
Plk was immunoprecipitated with Plk polyclonal antibody (Zymed) at 5 µg/ml and protein G Sepharose (Sigma) in the NP40 lysis buffer in the presence or absence of COOH-terminal Plk peptide (Ac-CLSSRSASNRLKAS-OH). Proteasome subunit components were immunoprecipitated with antibodies specific to individual proteasome subunits (Affiniti).

Transfection of HEK 293 Cells with HA-tagged Plk.
Human embryonic kidney 293 cells were transfected with 2.5 µg of DNA/10-cm plate according to the Qiagen SuperFect transfection protocol (25) . Thirty-six h after transfection, cells were synchronized with nocodazole to maximize the expression level of HA-Plk. Cells were lysed in the NP40 lysis buffer, and S100 was recovered. HA-tagged Plk was immunoprecipitated with a monoclonal antibody specific to the HA tag (BAbCo).

In Vitro Phosphorylation.
With immunoprecipitated samples, beads were washed twice in the kinase buffer [20 mM HEPES (pH 7.4), 150 mM KCl, 10 mM MgCl2, 1 mM EGTA, 0.5 mM DTT, 5 mM NaF, and 0.1 mM orthovanadate], and the kinase reaction was performed in 20 µl of kinase buffer containing 10 µCi [{gamma}-32P]ATP (3000 Ci/mmol; DuPont NEN) at 37°C for 1 h. The reaction was terminated by addition of 5x Laemmli SDS sample buffer and boiled for 5 min. For reimmunoprecipitation samples, the reaction mixture was boiled in 1% (w/v) SDS for 5 min and diluted in 1 ml of RIPA buffer [50 mM Tris (pH 7.4), 1% NP40, 1% deoxycholate, 0.1% SDS, 350 mM NaCl, 5 mM NaF, and 5 mM EGTA]. The supernatant was recovered for the second immunoprecipitation.

Purification of 20S Proteasomes.
20S proteasomes were purified by multiple chromatographic steps from asynchronous CA46 cells according to the procedure of Brown and Monaco (40) .

Monitoring of Proteasome Activity.
Proteasome activity was tested as described by Brown and Monaco (40) . Briefly, proteasome preparations were added to 100 µl of 100 mM Tris/acetate (pH 7.0) with 250 nM fluorogenic substrate Suc-Leu-Leu-Val-Tyr-AMC (CalBiochem). The reactions were carried out at 37°C for 30 min and terminated by the addition of 100 µl of ice-cold ethanol. Fluorescence was measured (excitation, 380 nm; emission, 460 nm) on a fluorometer (Turner Design).

To monitor proteasome activity during purification, 5 µl of each fraction were used. To test changes of proteasome activity after in vitro phosphorylation (Table 2A)Citation , the reaction mixture supernatant was recovered, combined with a 25-µl wash from the beads, and added to Tris/acetate buffer containing substrate.

To test proteasome activity in 293 cells, cells were lysed in NP40 buffer supplemented with phosphatase inhibitors (20 nM okadaic acid, 2 nM calyculin A) 36 h after transfection. 20S proteasomes were immunoprecipitated with antibodies against the "core" and {zeta} subunits. Immunoprecipitates were washed extensively in the NP40 buffer and then in Tris/acetate buffer, and proteolytic activities associated with the immunoprecipitates were detected as described above with Suc-Leu-Leu-Val-Tyr-AMC as substrate.

Expression of His6-Plk in Insect Cells.
The XhoI/StuI fragment (encoding amino acids 10–600) of the murine Plk cDNA was excised from pBlueScript-Th10 (41) and ligated to pAcHLT vector (Pharmingen) already digested with XhoI and StuI. pAcHLT-mPlk was cotransfected into SF9 insect cells with linearized BaculoGOLD virus DNA (Pharmingen). The culture medium containing virus-carrying recombinant Plk was amplified three times. His6-Plk was maximally expressed 72 h after infection. The recombinant protein His6-Plk was purified from insect cell lysates with ProBond nickel-chelating resin (Invitrogen) under native conditions. Solubility and kinase activity of the recombinant protein were tested. Some insect cells were treated with 1 µM okadaic acid for 4 h before harvest. To obtain M-phase lysate-activated His6-Plk, purified His6-Plk on beads was incubated with CA46 mitotic lysates with 1 mM ATP at 4°C or 37°C for 1 h.

Electrophoresis and Immunoblotting.
Proteins were separated on 10% SDS-PAGE and transferred to Immobilon-P (Millipore). Blots were blocked in 4% nonfat dry milk/TBST [10mM Tris-base (pH 7.4), 0.9% NaCl, 0.05% Tween 20], incubated in the primary antibody solution for 2 h, and rinsed, then incubated in the peroxidase-conjugated secondary antibody solution for 1 h. Blots were rinsed, and specific signals were detected with the Chemiluminescence Reagent (DuPont NEN).

Two-dimensional NEPHGE. The in vitro kinase reaction mixtures of 20S/His6-Plk was separated on a 6% acrylamide NEPHGE according to Farrar and Ferris (42) . The gel was then applied to the top of the second dimension gel, 12% SDS-PAGE. After the second dimension electrophoresis, the gels were transferred to Immobilon-P for autoradiography, followed by immunoblotting.

32P Metabolic Labeling.
HEK 293 cells were transfected 2 days before labeling. Cells were washed three times with P-DMEM (Life Technologies, Inc.), and then cultured for 3 h in P-DMEM supplemented with 10% phosphate-free fetal bovine serum and 0.5 mCi/ml [32P]orthophosphate (DuPont NEN). Cells were rinsed with cold P-DMEM three times and lysed in NP40 lysis buffer supplemented with 20 nM okadaic acid and 2 nM calyculin A. Clarified lysates were preabsorbed with protein G-Sepharose for 3 h at 4°C. Proteasomes were immunoprecipitated with pAb to "core" and mAb to {zeta} subunit. Reimmunoprecipitations with C9 and C8 mAb were carried out as above.

Acknowledgments

We thank Drs. Ira O. Daar and Chou-Chi Li for critical reading and valuable suggestions on the manuscript and Dr. Roger Moore of The Beckman Research Institute of The City of Hope (funding from NIH Cancer Center Support Grant CA33572) for providing technical service.

Footnotes

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This research was funded by the National Cancer Institute under Contract NO1CO56000 (to Science Applications International Corp.). Back

2 To whom requests for reprints should be addressed, at Biological Mechanisms Section, Laboratory of Leukocyte Biology, National Cancer Institute—Frederick Cancer Research and Development Center, Frederick, MD 21702. Phone: (301) 846-1429; Fax: (301) 846-6641; E-mail: Ferris{at}mail.ncifcrf.gov Back

3 The abbreviations used are: Plk, polo-like kinase; APC, anaphasepromoting complex; NEPHGE, nonequilibrating pH gel electrophoresis; P-DMEM, phosphate-free DMEM; mAb, monoclonal antibody. Back

Received for publication 9/ 6/00. Revision received 11/28/00. Accepted for publication 11/29/00.

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