Subcellular localization, modification and protein complex formation of the cdk-inhibitor p16 in Rb-functional and Rb-inactivated tumor cells

The cdk-inhibitor p16 is a tumor suppressor gene that is inactivated in many forms of cancer. Despite numerous studies, the exact mechanism of regulation of p16 has not been clarified, although the status of retinoblastoma (Rb) seems to be one important factor that influences the p16 expression. The specificity and validity of cytoplasmic localization of p16 observed in some tumors has further been questioned. Here, by subcellular fractionation of Rb-functional and Rb-inactivated cell lines, we show that p16 indeed is expressed in the cytoplasm as well as in the nucleus. Post translational modifications of p16 in different subcellular compartments as well as its capacity to form protein complexes were further delineated. Two dimensional gel electrophoresis showed that two forms of p16 appeared in the cytoplasm, while only one form was detected in the nucleus. Samples of basal cell carcinoma and squamous cell carcinoma of the skin with either functional or non-functional Rb also exhibited at least two forms of p16. In addition, cytoplasmic p16 bound cyclin dependent kinase (cdk)4/6, potentially indicating that p16 could have a function in the cytoplasm. (c) 2005 Wiley-Liss, Inc.