Proteolysis of Xenopus Cip-type CDK inhibitor, p16Xic2, is regulated by PCNA binding and CDK2 phosphorylation
© Zhu et al.; licensee BioMed Central Ltd. 2013
Received: 25 January 2013
Accepted: 17 April 2013
Published: 22 April 2013
Cell division is positively regulated by cyclin-dependent kinases (CDKs) partnered with cyclins and negatively regulated by CDK inhibitors. In the frog, Xenopus laevis, three types of CDK inhibitors have been described: p27Xic1 (Xic1) which shares sequence homology with both p21Cip1 and p27Kip1 from mammals, p16Xic2 (Xic2) which shares sequence homology with p21Cip1, and p17Xic3 (Xic3) which shares sequence homology with p27Kip1. While past studies have demonstrated that during DNA polymerase switching, Xic1 is targeted for protein turnover dependent upon DNA, Proliferating Cell Nuclear Antigen (PCNA), and the ubiquitin ligase CRL4Cdt2, little is known about the processes that regulate Xic2 or Xic3.
We used the Xenopus interphase egg extract as a model system to examine the regulation of Xic2 by proteolysis and phosphorylation.
Our studies indicated that following primer synthesis during the initiation of DNA replication, Xic2 is targeted for DNA- and PCNA-dependent ubiquitin-mediated proteolysis and that Cdt2 can promote Xic2 turnover. Additionally, during interphase, Xic2 is phosphorylated by CDK2 at Ser-98 and Ser-131 in a DNA-independent manner, inhibiting Xic2 turnover. In the presence of double-stranded DNA ends, Xic2 is also phosphorylated at Ser-78 and Ser-81 by a caffeine-sensitive kinase, but this phosphorylation does not alter Xic2 turnover. Conversely, in the presence or absence of DNA, Xic3 was stable in the Xenopus interphase egg extract and did not exhibit a shift indicative of phosphorylation.
During interphase, Xic2 is targeted for DNA- and PCNA-dependent proteolysis that is negatively regulated by CDK2 phosphorylation. During a response to DNA damage, Xic2 may be alternatively regulated by phosphorylation by a caffeine-sensitive kinase. Our studies suggest that the three types of Xenopus CDK inhibitors, Xic1, Xic2, and Xic3 appear to be uniquely regulated which may reflect their specialized roles during cell division or early development in the frog.
KeywordsXic2 Xenopus PCNA Phosphorylation Proteolysis CDK inhibitor
Low speed supernatant
High speed supernatant
Xenopus sperm chromatin
Polymerase chain reaction
Proliferating cell nuclear antigen
Sodium dodecyl sulfate
Polyacrylamide gel electrophoresis
Calf intestinal phosphatase
Rabbit reticulocyte lysate
In vitro translated.
The vertebrate cell cycle is positively regulated by cyclin-dependent kinases (CDKs) and negatively regulated by CDK inhibitors . Vertebrate CDK inhibitors of the Cip/Kip-type bind to and negatively regulate CDK2-cyclins E/A and the onset of DNA replication . Cip-type CDK inhibitors also bind to and negatively regulate the replication protein, Proliferating Cell Nuclear Antigen (PCNA) . Studies have indicated that mammalian Cip/Kip-type CDK inhibitors are frequently targeted for ubiquitin-mediated protein turnover during the G1 to S phase transition resulting in the activation of CDK2-cyclins and the progression into S phase [4–8].
In the frog, Xenopus laevis, three Cip/Kip-type CDK inhibitors have been identified that share sequence homology with mammalian p21Cip1 (p21), p27Kip1 (p27), and p57Kip2 (p57) [9–11]. p27Xic1/p28Kix1 (Xic1/Kix1) shares homology with all three of the mammalian Cip/Kip-type CDK inhibitors while p16Xic2 (Xic2) is more closely related to p21 and p17Xic3 (Xic3) is more closely related to p27 [9–11]. Developmental studies suggest that Xic1 is the only CDK inhibitor that is expressed in the early embryo and studies indicate that Xic1 is required for both the differentiation of nerve and muscles cells [11–18]. The expression of both Xic2 and Xic3 appears to be more tissue-specific in nature with Xic2 found in somites, the tail bud, lens, and the cement gland while Xic3 is expressed primarily in the central nervous system . While overexpression of Xic2 and Xic3 in the developing embryo results in an arrest in cell division due to an inhibition of CDK2 activity, little is known about the possible regulatory pathways that may control the activities of Xic2 or Xic3 .
Using the Xenopus interphase egg extract as a model biochemical system to study DNA replication initiation and CDK inhibitor regulation, studies have demonstrated that Xic1 is targeted for ubiquitination by the ubiquitin ligase, CRL4Cdt2, in a DNA- and PCNA-dependent manner during DNA polymerase switching resulting in its degradation [19–21]. In an effort to understand the possible molecular mechanisms that may regulate Xic2 and Xic3, we have taken a similar approach and used the Xenopus interphase egg extract as a biochemical model system to study Xic2 and Xic3. Our results suggest that while Xic3 appears to be stable in the Xenopus extract, Xic2 is targeted for ubiquitination and phosphorylation in the extract in a manner that is dependent upon specific DNA templates.
Xenopus Cip/Kip-type CDK inhibitors are differentially modified in the interphase egg extract
To examine Xic2 in a more physiological manner, we generated an antibody to Xic2 and immunoblotted the Xic2 protein in the Xenopus interphase egg extract and in Xenopus Tissue Culture (XTC) cells. We found that Xic2 was present at very low levels in the interphase egg extract following immunoprecipitation and immunoblotting (Figure 1D, left panel), while in XTC cells, Xic2 was readily detectable as a single protein band (Figure 1D, left panel). Moreover, we found that following ionizing irradiation (IR) of XTC cells, the expression of Xic2 was greatly increased and was easily detectable by direct immunoblotting (Figure 1D, right panel). This result suggests that Xic2 is not highly expressed in the early embryo, but becomes more highly expressed in somatic cells. Additionally, this result suggests that like mammalian p21 , Xic2 is highly induced following exposure to IR.
Xic2 is uniquely phosphorylated during the cell cycle and in response to single-stranded DNA
Xic2 turnover is dependent upon PCNA and PCNA binding
The addition of Cdt2, but not Skp2, promotes the turnover of Xic2
Xic2 phosphorylation by CDK2 inhibits its proteolysis during interphase
To directly test the role of CDK2 phosphorylation on Xic2 stability, we mutated the three possible CDK consensus sites within Xic2 individually and in combination to alanine to prevent phosphorylation (Figure 5B, C, D). Mutation of residue Thr-58 (T58) had little effect on Xic2 turnover, while mutation of residues Ser-98 (S98) or Ser-131 (S131) both resulted in more efficient proteolysis of Xic2 (Figure 5C). Additionally, mutation of S98 abolished phosphoform 1 of Xic2 suggesting that phosphorylation of Xic2 at residue 98 is responsible for the shifted phosphoform 1 of Xic2 in the interphase extract (Figure 5C). Simultaneous triple mutation of Xic2 at residues T58, S98, and S131 resulted in a Xic2 mutant that did not exhibit a shift and was efficiently degraded in the interphase extract compared to wildtype Xic2 (18% Xic2 remaining for the T58A, S98A, S131A mutant compared to 75% remaining for WT Xic2) (Figure 5D). We further generated glutamic acid mutations of Xic2 at residues S98 and S131 to mimic constitutive phosphorylation and tested these Xic2 mutants for turnover. Our studies indicated that the Xic2-S98E, S131E double mutant and the S98E and S131E single point mutants were all degraded similarly or less efficiently than the wildtype Xic2 that is phosphorylated in the extract (Xic2 WT and the E mutants are degraded to approximately 50% of Xic2 remaining, Figure 5E, versus 18% for the Xic2 triple alanine mutant that is not phosphorylated, Figure 5D). This result is consistent with our hypothesis that Xic2 phosphorylation in the extract inhibits its turnover. Taken together, these studies suggest that CDK2-dependent phosphorylation of Xic2 at residues S98 and S131 negatively regulates its ubiquitination and degradation during interphase.
Xic2 is hyperphosphorylated at residues S78/S81 in a manner dependent upon single-stranded DNA and a caffeine-sensitive kinase
An examination of the potential sites targeted for phosphorylation of Xic2 in the presence of ssDNA revealed two residues at S78 and S81 that matched the S/T-Q consensus site for ATM-ATR-like kinases (Figure 6D, top). Mutation of these two residues to alanine (S78A, S81A) completely eliminated the Xic2 phosphoforms 2 in the presence of ssDNA compared to wildtype Xic2 (Figure 6D, bottom, samples for “S78A, S81A” and “S78A, S81A, S98A”). Phosphorylation of Xic2 by the caffeine-sensitive kinase in the presence of ssDNA does not appear to be dependent upon prior or concurrent phosphorylation of Xic2 by CDK2-cyclin as shown by the shift of a Xic2-S98A mutant (Figure 6D) and by the ability of Xic2 to be phosphorylated in an ssDNA-dependent manner in the presence of roscovitine (data not shown). This suggests that the interphase phosphorylation of Xic2 by CDK2 and the caffeine-sensitive kinase function along independent pathways.
Our studies suggest that like mammalian p21, Xic2 protein levels appear to be induced upon treatment with IR . This result suggests that in addition to a role in development, Xic2 may also play a role in a DNA damage checkpoint with Xic2 levels increasing during DNA damage, similar to Xic1, to presumably halt entry into S phase to allow DNA repair to occur (Figure 7B, bottom right) . Consistent with this hypothesis, Xic2 appears to be phosphorylated by a caffeine-sensitive kinase in the presence of double-stranded DNA ends, a signal shown to activate a DNA damage checkpoint in the interphase egg extract [32, 33] (Figure 7A). Under the same conditions, XCds1 (Xenopus Chk2) has been shown to be phosphorylated and this phosphorylation is inhibited by a block to DNA replication and by caffeine suggesting Xic2 may be modulated by the same checkpoint pathway as XCds1 . How the phosphorylation of Xic2 at residues S78 and S81 may influence its CDK2 inhibitory activity or binding to CDK2 and PCNA are currently under further investigation.
The fact that Xic2 is readily ubiquitinated and degraded in the interphase egg extract when CDK2 phosphorylation is inhibited suggests that the ubiquitination machinery that targets Xic2 for degradation is present in the egg. Our past studies of the Xenopus interphase egg extract suggest that the CRL4Cdt2 ubiquitin ligase is active in the extract while the SCFSkp2 ubiquitin ligase is not due to very low expression of Skp2 in the egg [20, 24]. Our results demonstrating the dependence of Xic2 proteolysis on PCNA and DNA, the timing of Xic2 ubiquitination and degradation, and the ability of Cdt2 to promote Xic2 turnover are all consistent with CRL4Cdt2 being the ubiquitin ligase of Xic2 in the egg extract. It is then somewhat puzzling that Xic2 does not readily bind to Cdt2 under conditions that efficiently support Xic1 and Cdt2 binding. Similar to p21 and Xic1, Xic2 bears the “specialized PIP box” described by Havens and Walters [29, 35] which contains a PIP box that binds PCNA with high affinity (contains the TD motif) followed by a basic residue at the +4 position relative to the PIP box. Our findings suggest that Xic2 may more closely resemble the replication protein and CRL4Cdt2 substrate, Cdt1, in requiring the presence of DNA to appreciably bind to Cdt2. However, unlike Cdt1, Xic2 readily binds PCNA in the absence of DNA. It is also possible that Xic2 is only indirectly targeted by Cdt2 or that Xic2 is additionally targeted by an alternative E3 in the egg extract, although our studies indicated that the addition of Skp2 did not promote Xic2 turnover. It is interesting to note that sequence analyses revealed a KEN box present in both Xic2 (83KENQCQD89) and Xic1 (138KENAEKI144) suggesting that Xic2 and Xic1 may be targeted for ubiquitination by the ubiquitin ligase, APCCdh1. However, Cdh1 expression in the Xenopus egg is very low and it is not appreciably expressed until after gastrulation .
Using the Xenopus model extract system, we have evidence to support that Xic2 is phosphorylated in a CDK2 dependent manner, most likely by CDK2-cyclin E, the predominant CDK activity in the interphase egg extract  (Figure 7A). The phosphorylation of Xic2 at residues S98 and S131 appear to equally inhibit the ubiquitination and degradation of Xic2 in the extract. The inhibition of Xic2 turnover by CDK2 phosphorylation may be due to a change in the cell localization of Xic2 upon phosphorylation although this is unlikely to be the predominant contributing factor since our degradation studies using the HSS extract which does not contain nuclear membrane precursors and does not support nuclei formation  still showed that CDK2 phosphorylated Xic2 was inhibited for degradation. Alternatively, Xic2 phosphorylation by CDK2 may influence the binding of Xic2 to PCNA, DNA, or the ubiquitination machinery. The finding that CDK2 phosphorylation of Xic2 inhibits its PCNA-dependent turnover suggests that Xic2 phosphorylation may be an important regulator of Xic2 stability and function.
In this study, we provide the first biochemical examination of the regulation of the Xenopus CDK inhibitors, p16Xic2 and p17Xic3, using the egg extract model system. Our studies indicate that Xic2 is targeted for DNA- and PCNA-dependent ubiquitination and degradation in the interphase egg extract and that this turnover of Xic2 is promoted by Cdt2 and inhibited by CDK2-dependent phosphorylation of Xic2 at residues Ser-98 and Ser-131. Additionally, it appears that during conditions mimicking a DNA damage checkpoint, Xic2 is targeted for phosphorylation by a caffeine-sensitive kinase at residues Ser-78 and Ser-81, although the consequence of this phosphorylation is still unclear. Xic3 appears to be stable in the interphase egg extract in the presence or absence of DNA.
In their initial discovery of Xic2, Daniels et al.  described Xic2 as an ortholog of mammalian p21 which is known to be transcriptionally induced by p53 upon DNA damage. p21 has also been shown to be a substrate of both CRL4Cdt2 and SCFSkp2[38–40]. The RNA expression pattern of Xic2 in somites, the tail bud, the lens, and the cement gland suggest that Xic2 protein is expressed during late embryonic development , but how Xic2 protein may be regulated by proteolysis during development remains unknown. It will be important to study how Xic2 may be regulated by PCNA, CRL4Cdt2, or CDK2 during developmental patterning. It will also be necessary to study Xic1, Xic2, and Xic3 and their regulators in the context of the developing embryo and the somatic cell to fully understand how these three Xenopus CDK inhibitors mediate the events of early development and cell cycle control in the frog.
Preparation of Xenopus extracts and demembranated sperm chromatin
Xenopus interphase extracts [low speed supernatant (LSS) and high speed supernatant (HSS)] , stable Δ90 mitotic extract (LSS supplemented with Δ90 non-degradable cyclin B), and demembranated Xenopus sperm chromatin (XSC) [19, 41, 42] were prepared as previously described. All studies involving animals were conducted according to the rules established by the Universities of Federation for Animal Welfare, the World Society for the Protection of Animals Working Party, and the American Veterinary Medical Association. This work was approved by the Institutional Animal Care and Use Committee of the University of Texas Health Science Center at San Antonio which is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care under protocols 99045I-04-06-A and 11073x.
Xenopus Tissue Culture (XTC) cells were propagated at room temperature in L-15 medium with L-glutamine (Sigma-Aldrich) supplemented with 10% fetal bovine serum (Gibco-BRL) and 50 ug/ml of penicillin-streptomycin (Gibco-BRL). Cells were irradiated with 10 Gy using a 137Cesium source Mark I Model 68A irradiator and harvested 4 and 8 hours later in RIPA buffer (10 mM Tris-Cl, pH 8.0, 1mM EDTA, 0.15 M NaCl, 1% Np-40 and 1% Sodium Deoxycholate) containing protease inhibitors (Sigma, P8340).
Generation of Xic2 mutants and other constructs
All point mutants of Xic2 in pCS2+ were generated by using pCS2+-Xic2 as the template and the QuickChange™ Site-Directed Mutagenesis Kit (Stratagene) followed by DNA sequencing to confirm the mutagenesis. Mutagenesis primer sequences are available upon request. pGEX4T-Xic2 was generated by subcloning a PCR fragment of Xic2 using pCS2+-Xic2 as the template into the BamHI and SalI restriction sites of pGEX4T-1.
Production of Xic2 antibody and other antibodies
GST-Xic2 expressed in BL21Star (DE3) was purified using Glutathione-Sepharose 4B beads (Amersham Biosciences) according to the manufacturer’s instructions. Rabbit polyclonal antibody against GST-Xic2 was generated by the University of Texas Health Science Center at San Antonio SACI antibody core facility. Anti-PCNA mouse monoclonal antibody was purchased from Santa Cruz (P-10) and anti-GST-XCyclin E antibody was a gift from Peter K. Jackson and Marc W. Kirschner.
In vitro transcription and translation
In vitro transcription and translation reactions were performed using the SP6 TNT coupled reticulocyte lysate system (Promega) and 35S-methionine from New England Nuclear.
Dephosphorylation and inhibition assays
Dephosphorylation of protein samples was performed using lambda phosphatase (λ-PPase) (New England Biolab, P0753S). Reactions were incubated at 30°C for 30 min in the presence of 80 or 200 units of λ-PPase, terminated by the addition of protein loading dye, and analyzed by SDS-PAGE and phosphorimager. Roscovitine (A. G. Scientific Inc, R-1016) was used as previously described to inhibit CDK2 activity . Caffeine (Sigma) was used at a final concentration of 10 mM from fresh stocks of 75 mM in XB- (100 mM KCl, 1 mM MgCl2, 0.1 mM CaCl2, 10 mM HEPES pH 7.7).
GST pull down assays, immunoblotting, and immunoprecipiation
GST pull down assays using egg extract and immunoblotting were performed as previously described except the binding reactions were conducted at 4°C for 2 hours [21, 22]. Rabbit serum against Xic2 was used to immunoprecipitate and immunoblot Xic2 in extracts. Anti-cyclin E antibody was used to immunoprecipiate cyclin E from extracts while anti-PCNA antibody was used for immunoblots. Normal rabbit serum was used as a control for immunoprecipitations.
Degradation assay, nuclei spin down assay, and phosphorylation shift assay
Degradation assays were performed as previously described  with the following modifications. Proteins labeled with 35S-methionine were added to extracts at a final dilution of 1:15 in the presence or absence of 10 ng/ul demembranated XSC or ΦX174 single-stranded DNA (New England Biolab, N3023S). The reactions were analyzed by PhosphorImager and quantitation was performed using ImageQuant™ software (Molecular Dynamics). The percentage of protein remaining for each sample was determined by normalizing the amount of protein at the 0 hr time point to 100%. Nuclei spin down assays were performed as previously described [23, 24] with the following minor modifications. LSS was incubated with ubiquitin (3 ug/ul), 35S-methionine labeled Xic2 at a final dilution of 1:15, and XSC (10 ng/ul) for 90 min at 23°C. When indicated, methyl ubiquitin was added to a final concentration of 3 ug/ul. Ubiquitin (Sigma) was methylated as previously described . In vitro translated Chk2 (XCds1-pCS2+) or Xic2 was added to HSS and incubated for 0 to 3 hours in the presence or absence of double-stranded DNA ends that were generated from annealed oligonucleotides .
We are grateful to all the past and present members of the Yew lab for helpful discussions, in particular, Li-Chiou Chuang and Horng-Ru Lin; Shin-ichi Ohnuma for kindly providing pCS2+-Xic2 and pCS2+-Xic3 plasmids; Peter K. Jackson and Marc W. Kirschner for anti-GST-XCyclin E antibodies; Li-Chiou Chuang for purified His6-XPCNA and GST-human p27; Ethan Lee for the MBP-Δ90 cyclin B1 expression construct; Jason Spaeth for GST-Xic1(161–190) bacterial lysate; Todd Stukenberg for XTC cells and culture conditions; The San Antonio Cancer Institute Cancer Center Support Grant P30 CA54174 for Xic2 antibody production; Carlos R. Herrera for MBP-Δ90 Cyclin B protein; and Michael J. Parker and Carlos R. Herrera for excellent technical assistance. This work was supported by the National Institute of Health (RO1-GM066226) (PRY).
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