Monoclonal antibodies to activated CDK4: use to investigate normal and cancerous cell cycle regulation and involvement of phosphorylations of p21 and p27

Cyclin-dependent kinase 4 (CDK4) is a master integrator that couples mitogenic/oncogenic signaling with the cell division cycle. It is deregulated in most cancers and inhibitors of CDK4 have become standard of care drugs for metastatic estrogen-receptor positive breast cancers and are being evaluated in a variety of other cancers. We previously characterized the T-loop phosphorylation at T172 of CDK4 as the highly regulated step that determines the activity of cyclin D-CDK4 complexes. Moreover we demonstrated that the highly variable detection of T172-phosphorylated CDK4 signals the presence or absence of the active CDK4 targeted by the CDK4/6 inhibitory drugs, which predicts the tumor cell sensitivity to these drugs including palbociclib. To date, the phosphorylation of CDK4 has been very poorly studied because only few biochemical techniques and reagents are available for it. In addition, the available ones including 2D-IEF separation of CDK4 modified forms are considered too tedious. The present report describes the generation, selection and characterization of the first monoclonal antibodies that specifically recognize the active CDK4 phosphorylated on its T172 residue. One key to this success was the immunization with a long phosphopeptide corresponding to the complete activation segment of CDK4. These monoclonal antibodies specifically recognize T172-phosphorylated CDK4 in a variety of assays, including western blotting, immunoprecipitation and, as a capture antibody, a sensitive ELISA from cell lysates. The specific immunoprecipitation of T172-phosphorylated CDK4 allowed to clarify the involvement of phosphorylations of co-immunoprecipitated p21 and p27, showing a privileged interaction of T172-phosphorylated CDK4 with S130-phosphorylated p21 and S10-phosphorylated p27.
<b>Abbreviations</b>:
2D: two-dimensional; CAK: CDK-activating kinase; CDK: cyclin-dependent kinase; HAT: Hypoxanthine-Aminopterin-Thymidine; FBS: fetal bovine serum; IP: immunoprecipitation; ID: immunodetection; mAb: monoclonal antibody; PAGE: polyacrylamide gel electrophoresis; PBS: phosphate buffer saline; pRb: retinoblastoma susceptibility protein; SDS: sodium dodecyl sulfate; DTT: dithiotreitol; TET: tetracyclin repressor; Avi: Avi tag; TEV: tobacco etch virus cleavage site; EGFP: enhanced green fluorescent protein; BirA: bifunctional protein biotin ligase BirA; IRES: internal ribosome entry site; HIS: poly-HIS purification tag; DELFIA: dissociation-enhanced lanthanide fluorescent immunoassay; 3-MBPP1: 1-(1,1-dimethylethyl)-3[(3-methylphenyl) methyl]-1H-pyrazolo[3,4-d] pyrimidin-4-amine; BSA: bovine serum albumin; ECL: Enhanced chemiluminescence

细胞周期蛋白依赖性激酶4（cyclin-dependent kinase 4，CDK4）是将促有丝分裂/致癌信号与细胞分裂周期相偶联的核心整合因子。该蛋白在大多数癌症中存在表达或功能失调，CDK4抑制剂已成为转移性雌激素受体阳性乳腺癌的标准治疗药物，目前正针对多种其他癌症开展临床评估。
我们此前已明确，CDK4 T172位点的T环磷酸化是决定细胞周期蛋白D-CDK4复合物活性的高度调控步骤。此外，我们证实，T172磷酸化CDK4的信号检测差异极大，这一现象可反映CDK4/6抑制性药物靶向的活性CDK4是否存在，能够预测肿瘤细胞对包括帕博西尼（palbociclib）在内的此类药物的敏感性。
迄今为止，CDK4的磷酸化研究仍十分匮乏，原因在于可用的生化技术与试剂极少。此外，现有技术手段（如CDK4修饰形式的二维等电聚焦（2D-IEF）分离）被认为过于繁琐。
本研究报道了首款特异性识别T172位点磷酸化活性CDK4的单克隆抗体的制备、筛选与表征。此次成功的关键之一，是使用对应CDK4完整激活环的长磷酸肽进行免疫接种。
这些单克隆抗体可通过多种实验技术特异性识别T172磷酸化CDK4，包括蛋白质免疫印迹、免疫沉淀，以及作为捕获抗体用于细胞裂解液中高灵敏度的酶联免疫吸附实验（ELISA）。
对T172磷酸化CDK4的特异性免疫沉淀，帮助阐明了共沉淀的p21与p27的磷酸化修饰参与情况，结果显示T172磷酸化CDK4与S130磷酸化p21、S10磷酸化p27存在优先相互作用。
<b>缩写说明</b>：
2D：二维（two-dimensional）；CAK：CDK激活激酶（CDK-activating kinase）；CDK：细胞周期蛋白依赖性激酶（cyclin-dependent kinase）；HAT：次黄嘌呤-氨基蝶呤-胸腺嘧啶（Hypoxanthine-Aminopterin-Thymidine）；FBS：胎牛血清（fetal bovine serum）；IP：免疫沉淀（immunoprecipitation）；ID：免疫检测（immunodetection）；mAb：单克隆抗体（monoclonal antibody）；PAGE：聚丙烯酰胺凝胶电泳（polyacrylamide gel electrophoresis）；PBS：磷酸盐缓冲液（phosphate buffer saline）；pRb：视网膜母细胞瘤易感蛋白（retinoblastoma susceptibility protein）；SDS：十二烷基硫酸钠（sodium dodecyl sulfate）；DTT：二硫苏糖醇（dithiotreitol）；TET：四环素阻遏蛋白（tetracyclin repressor）；Avi：Avi标签（Avi tag）；TEV：烟草蚀刻病毒切割位点（tobacco etch virus cleavage site）；EGFP：增强型绿色荧光蛋白（enhanced green fluorescent protein）；BirA：双功能蛋白生物素连接酶BirA（bifunctional protein biotin ligase BirA）；IRES：内部核糖体进入位点（internal ribosome entry site）；HIS：多组氨酸纯化标签（poly-HIS purification tag）；DELFIA：解离增强镧系荧光免疫分析法（dissociation-enhanced lanthanide fluorescent immunoassay）；3-MBPP1：1-(1,1-二甲基乙基)-3-[(3-甲基苯基)甲基]-1H-吡唑并[3,4-d]嘧啶-4-胺（1-(1,1-dimethylethyl)-3[(3-methylphenyl) methyl]-1H-pyrazolo[3,4-d] pyrimidin-4-amine）；BSA：牛血清白蛋白（bovine serum albumin）；ECL：增强化学发光法（Enhanced chemiluminescence）