Identication and Verication of Cdk5 Phosphorylated Deubiquitinating Enzyme BRCC3

Background: It is known that the expression of the deubiquitinating enzyme BRCA1-BRCA2-containing complex subunit 3(BRCC3) is increased, as well as Cyclin-dependent protein kinase 5(Cdk5) in Parkinson’s Disease(PD), and both of them to be involved in neuroinammatory response. But the regulatory mechanism of Cdk5 on the post-translational modication of BRCC3 remains unclear. This study aimed to investigate the phosphorylation of Cdk5 on the BRCC3. Methods and Results: Cdk5 phosphorylation of BRCC3 was predicted by GPS5.0 software. His-BRCC3 plasmid was constructed by cloning the BRCC3 gene into pGEX-6P-1 vector, and then His-BRCC3 fusion protein was induced by IPTG and puried by His-tag purication agarose. The BRCC3 fusion protein was reacted with Cdk5 in vitro, and the phosphorylation was detected by mass spectrometry and Western blot. The results showed that multiple phosphorylation sites were predicted by GPS5.0, and the His-BRCC3 fusion protein was successfully induced and puried. In vitro kinase assay, Western blot and mass spectrometry identied that Cdk5 can phosphorylate BRCC3. Conclusions: It has been demonstrated that the protein kinase Cdk5 can phosphorylate the deubiquitinating enzyme BRCC3 in vitro, which provide evidences for further study on the mechanism of neurodegeneration. PD[5]. To gain further insight into the relationship between Cdk5 and BRCC36, we predicted the phosphorylation sites of Cdk5 modied BRCC3 using GPS5.0 software. And phosphorylation mass spectrometry indicated that BRCC36 was really the substrate of Cdk5 kinase. Besides this, we also conrmed that BRCC3 can be phosphorylated by Cdk5 using Western blot. Our further analyses of these suggest that Cdk5 is an upstream kinase of BRCC3 in vitro. The study provides a strong basis for further exploring the molecular mechanism of Cdk5-mediated BRCC3 involvement in inammation in PD.


Introduction
Phosphorylation, methylation, ubiquitination, acetylation and glycosylation are conventional posttranslational modi cations of proteins. Phosphorylation often occurs during development of some diseases and is an important regulatory mode in prokaryotes and eukaryotes [1]. This process is completed by a series of kinase reactions in the organism, and plays a signi cant role in enzyme activities and cascade reactions. Proteins undergo posttranslational modi cation during synthesis. Some speci c modi cations are closely related to certain diseases, and improper modi cations may cause dysfunction of the body.
Cyclin-dependent kinase 5 (Cdk5), an important member of the Cdk family, is a proline-dependent serine/threonine protein kinase. As a protein kinase, many substrate proteins can be phosphorylated by Cdk5. These phosphorylation processes are involved in the molecular mechanism of disease and a variety of cellular signaling pathways, such as apoptosis, autophagy, DNA damage, etc. Cdk5 is highly expressed in the nervous system and is involved in neuronal differentiation, survival and synaptic occurrence, which is associated with the development of neurodegenerative diseases (eg. Parkinson's disease (PD), Alzheimer's disease (AD)) [2][3][4]. Cdk5 protein kinase alone does not have enzymatic activity, and it needs to combine with the companion cytokines cyclins P35 or P39 to form a complex. P35 can be spliced by speci c proteases to form P25, which changes the characteristics of Cdk5 and then participates in neurodegenerative diseases through phosphorylation [5].
Mouse-BRCC3 and its human homolog BRCC36, is a component of the BRCA1-A complex, which widely expressed in different tissues such as brain, muscle, kidney, etc. and regulates the abundance of Lys63-linked ubiquitin chains [6]. It has many connections with cell signal transduction, cell cycle regulation, DNA damage repair [7], and NF-kB signaling pathway [8,9]. Studies have shown that BRCC3 can regulate the activity of NLRP3 in ammation through deubiquitylation [5,10]. The activation signal can promote the deubiquitylation of NLRP3 through the lysine-speci c deubiquitinating enzyme BRCC3 and then activate NLRP3 in ammation [5,10]. The ubiquitin-dependent mechanism contributes to the development of potential inhibitor and activator therapeutic target drugs for in ammatory diseases.
We and others showed previously that Cdk5/P25 involved in the development of Parkinson's disease through phosphorylation [11][12][13][14]. Especially, Cdk5 can regulate the expression of BRCC3 in PD [5]. To gain further insight into the relationship between Cdk5 and BRCC36, we predicted the phosphorylation sites of Cdk5 modi ed BRCC3 using GPS5.0 software. And phosphorylation mass spectrometry indicated that BRCC36 was really the substrate of Cdk5 kinase.
Besides this, we also con rmed that BRCC3 can be phosphorylated by Cdk5 using Western blot. Our further analyses of these suggest that Cdk5 is an upstream kinase of BRCC3 in vitro. The study provides a strong basis for further exploring the molecular mechanism of Cdk5-mediated BRCC3 involvement in in ammation in PD.

Target gene ampli cation and plasmid recombination
The plasmid pEGFP-BRCC3 generated previously by our research team was invoked as DNA template, and the target fragment was ampli ed by RT-PCR. The PCR product was run electrophoresis with 1% agarose gel. The target DNA gel was harvested according to the molecular weight of BRCC3 and extracted with DNA gel extraction kit. In the presence of T4 ligase, the puri ed DNA product of BRCC3 was ligated at room temperature for 1h with the pGEX-6P-1 vector which digested by BamH I and Xhol I. The ligation product was transformed into DH5α competent cells and monoclonal strains were cultured. The possible recombinant bacterial colony was randomly picked, extracted and then identi ed as positive colony by double enzyme digestion. The suspected positive plasmid was sent to Sangon Biotech (Shanghai, Co., Ltd) for sequencing to con rm the successful recombination.

Induction and puri cation of His-BRCC3 fusion protein
The pGEX-6P-1-BRCC3-His construct was transformed into BL21 competent cells, and cultured in LB medium for 14-16 h to observe the growth of the clone.
Monoclones were selected and shaken at 37℃ for 16-18 h. Then some bacterial suspension was taken and added into 2×YTA medium (peptone 1.6g, yeast 0.8g, NaCl0.5g, ddH2O supplemented to 100 mL) at a ratio of 1:100, and continued shaking at 37℃ until OD600 to 0.8. At this time, IPTG (0.5 mM) was added into the bacterial suspension and incubated at 37℃. After 4h, the bacterial precipitation was collected by centrifugation. The supernatant was resuspended with lysis buffer (50mM NaH2PO4, 300mM NaCl, 10mM imidazole) and treated with ultrasonic homogenizer. Ni-NTA His-tag puri cation agarose was used to combine His-BRCC3 target protein according to the manufacture's instruction, then washed with washing buffer (50mM NaH2PO4, 300mM NaCl, 20mM imidazole), and His-BRCC3 protein was eluted with elution buffer (50mM NaH2PO4, 300mM NaCl, 250mM imidazole). Finally, SDS-PAGE gel was stained with Coomassie brilliant blue R250 to identify the induced His-BRCC3 fusion protein.

Kinase reaction in vitro
For in vitro kinase assays, active Cdk5/p25 complex protein was incubated with puri ed recombinant His-BRCC3 fusion protein in the kinase reaction buffer (8 mM MOPS/NaOH, 200 nM EDTA) plus 20 μM ATP at 30°C for 30 min. The reaction was stopped by adding SDT buffer (4%(w/v)SDS, 100mM Tris/HCl, 1Mm DTT, pH 7.6). The most of reaction product was analyzed by MALDI-MS/MS. The rest of it was boiled at 95°C for 5 min. The phosphorylation of substrate then was detected by autoradiography with phospho-Ser/Thr-proline antibody (1:1,000) following SDS-PAGE analysis. And total His-tagged proteins were re-probed with anti-His antibody (1:5,000).

Phosphorylation mass spectrometry
The sample of phosphorylated products were analyzed by Shanghai Applied Protein Technology Co. Ltd using LC-MS/MS(Nanolc-QE). Brie y, the chromatographic column was balanced with 95% liquid (solution of 0.1% formic acid), then the sample was loaded from the automatic sampler to the TRAP column for 1 hour. For mass spectral data collection, 20 fragment maps (MS2 scan) were collected after each full scan according to the mass charge ratio of polypeptides. The raw le of mass spectrometry test was retrieved from the database (R20190100160_ZJK.fasta) using Mascot2.2 software, and the identi ed protein results were obtained.

Prediction of BRCC3 phosphorylation by Cdk5
In order to know that Cdk5 may be an upstream kinase of BRCC3, phosphorylation prediction software GPS5.0 was used to predict the phosphorylation between BRCC3 and Cdk5. The result showed that BRCC3 was the phosphorylated substrate of Cdk5. Five phosphorylation sites (Ser16, Ser28, Ser83, Ser93, Ser233) were predicted by GPS5.0 software (Table 1). Ser93, the highest score (16.846) among them, was the most likely phosphorylation site. Moreover, amino acid sequences of BRCC3 protein from human, mouse and rat were compared by DNAMAN software. The result showed that BRCC3 had a high conservatism and homology of 96.33% among different species of mammals (Fig. 1a, 1b).
pGEX-6P-1-BRCC3-His plasmid construction and His-BRCC3 fusion protein expression To further verify the phosphorylation effect of Cdk5 on BRCC3, pGEX-6P-1-BRCC3 prokaryotic plasmid was constructed. The full-length target gene BRCC3 (876bp) was inserted into the pGEX-6P-1 vector (4984bp). The recombinant pGEX-6P-1-BRCC3 plasmid was detected through enzyme digestion of BamHI and XholI. The agarose gel electrophoresis results showed two DNA bands around 5000bp and 900bp, which were identi ed as pGEX-6P-1 vector and target fragment BRCC3 respectively (Fig. 2a). Moreover, DNA sequencing was performed by Sangon Biotech (Shanghai) Co., Ltd. The sequence of the recombinant plasmid was exactly matched with the target sequence, which further demonstrated that pGEX-6P-1-BRCC3 plasmid was successfully constructed (Fig. 2b). Next, the recombinant His-BRCC3 construct was induced by IPTG and the fusion protein was identi ed by staining SDS-PAGE gel with Coomassie brilliant blue. The results showed that His-BRCC3 fusion protein was successfully expressed, and its molecular weight was about 36kD (Fig. 2c).
The protein sample of phosphorylation product was analyzed using LC-MS/MS(nanoLC-QE). After getting the information of pep-tides, LC-MS/MS data were searched by Mascot mass spectrometry software to obtain the qualitative identi cation information of the target protein peptide molecules. The results of mass spectrometry showed that both BRCC3 and Cdk5 proteins were identi ed, and BRCC3 protein could be modi ed by oxidation and phosphorylation (Supplementary Table1). Eight phosphorylation sites (Ser227 Thr229 Ser233 Thr235 Ser241 Thr244, Ser249 and Ser252) were screened out from 11 nonredundant phosphorylated peptides (Table2 and Supplementary Table1).

Identi cation of Phosphorylation with Western blot
To con rm the reliability of the mass spectrometry data, the product of kinase assays in vitro was veri ed for phosphorylation using Western blot. Detection of BRCC3 with Phospho-Ser/Thr-proline antibody also indicated that BRCC3 could be phosphorylated by Cdk5 (Fig. 3). These results indicate that BRCC3 was a substrate of Cdk5 in vitro, and the information obtained by using MS was reliable and might provide guidance for further study of the roles of these two proteins in disease.

Discussion
Since Cdk5 and BRCC3 expression were elevated in Parkinson's disease [5,14], we speculated that Cdk5 might regulate the occurrence and progression of PD through phosphorylation of BRCC3. In order to prove the effect of Cdk5 on the deubiquitinating enzyme BRCC3, GPS5.0 software was used in this study to predict that Cdk5 might be the upstream kinase of BRCC3. The recombinant plasmid pGEX-6P-1-His-BRCC3 was constructed and successfully induced the His-BRCC3 fusion protein. After in vitro kinase reaction, the results of phosphorylation mass spectrometry and Western blot analysis showed that Cdk5 could phosphorylate BRCC3. This study provides a new perspective on the molecular mechanism of neurodevelopment and neurodegenerative diseases.
Cdk5 is a proline-dependent protein kinase that can phosphorylate serine and threonine residues, and its substrates contain (S/T) Px amino acid [15]. Qi et al.
[16] found connexin 43 (CX43) protein regulated neuron migration and localization through astrocyte-neuron interactions during the early embryonic development of neurons. When Cdk5 phosphorylates CX43 at Ser279 and Ser282, the membrane localization and degradation of CX43 were modulated during neuron differentiation [16]. Moreover, Cdk5 also participates in the course of neurodegenerative disease by phosphorylating a large number of substrates, leading to their activation or inactivation. Wang et al. [17] found that Cdk5 phosphorylates Ser516 of GP78 in Parkinson's disease via the ubiquitin-proteasome pathway in vitro and in vivo, suggesting that the Cdk5-GP78 pathway provides a new pathway for neuroprotection in midbrain dopaminergic neurons. In Alzheimer's disease, Cdk5 hyperphosphorylates the Tau protein, causing a conformational change in the Tau protein that misfolds to form a double helix lament, eventually forming nerve ber tangles [18]. Besides, Satow et al. [19] found that Cdk5 could regulate the expression of TPX2 by phosphorylating TPX2 Ser486, thereby promoting the proliferation and tumorigenicity of liver cells. In addition, Cdk5 can also regulate the molecular process of disease through phosphorylation of FAK, DRP1, PAK1, β-catenin, SRC, MAP1B, Nudel, Munc18, amphyphysin, signal molecules DAB1, Nudel, and so on [20]. Therefore, Cdk5 phosphorylation of BRCC3 in this study may also play an important pathway regulation role in nervous system diseases.
The deubiquitinating enzyme BRCC36 is a catalytic subunit of two multiprotein complexes, BRCA1-A and BRISC. In which, BRCA1-A protects genomic integrity by regulating the selection of DNA repair pathways, while BRISC serves cellular stress response and immune signaling function. BRCC3 promotes the repair of DNA double-strand breaks by removing H2AX (histone H2A)[6] and RAP80 (receptor associated protein 80) [21] ubiquitination, and participates in the repair of DNA cross-linking damage by removing the FANCG Fanconi anemia complementation group G ubiquitination. BRCC3 also plays a role in cell cycle regulation by removing TNKs tankyrase and NUMA nuclear mitotic appratus ubiquitination [22,23]. Studies have shown that BRCC3 can regulate the activation of NLRP3 in ammasome through deubiquitination, while deubiquitinating enzyme inhibitor G5 can inhibit the deubiquitination of NLRP3 in ammasome activation and the secretion of IL-1β [10]. Consistent with this, our previous studies also proved that BRCC3 was involved in the activation of NLRP3 in ammasome in Parkinson's Disease [5].

Conclusion
In the present study, the phosphorylation of BRCC3 by Cdk5 was identi ed in vitro. Although the sites predicted by GPS5.0 was not completely consistent with the LC-MS/MS, the mechanisms of these kinase-substrate interactions need further investigation. The next study will focus on identifying the speci c sites where BRCC3 is phosphorylated by Cdk5 and further prove its regulatory role in PD, so as to provide new support for the prevention and treatment of PD.     Identi cation of Phosphorylation with Western blot His-BRCC3 fusion protein reacted with active Cdk5/p25 kinase in vitro. The reaction product was performed by Western blot the phosphorylation of BRCC3 was detected with phospho-Ser/Thr-proline antibody, and the expression of total BRCC3 was detected with His-tag antibody.