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

Abstract

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 Rbfunctional 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. ' 2005 Wiley-Liss, Inc.Key words: p16; retinoblastoma gene product; two-dimensional gel electrophoresis Progression of a cell through the cell cycle is tightly regulated by several control mechanisms involving positive and negative regulators of the cell cycle. Uncontrolled regulation of these control mechanisms leads to dysfunctional cell cycle control, uncontrolled cell division and eventually tumor formation. 1 Central in the late G1 phase checkpoint is the retinoblastoma (Rb) pathway. In G1 phase, Rb binds to transcription factors such as E2Fs, preventing genes necessary for S-phase entry to be transcribed. Upon mitogen stimulation, there is an upregulation of cyclin D which binds to cyclin dependent kinases (cdks) 4/6 and the complex initiates the phosphorylation of Rb, which becomes inactive. This leads to release of E2Fs and transcription of genes that are needed for the cell to continue into S-phase. 2 The cdk-inhibitor p16, with the major function to block the cell cycle, is a tumor suppressor gene that is inactivated in many cancer forms. 3 When p16 in the G1-phase of the cell cycle binds to cdk4/6, the active cyclin Dcdk4/6 complex cannot be formed and phosphorylation of Rb is thereby prevented causing inhibition of the cell cycle. 4,5 Small homozygous deletions, point mutations and methylation of the promoter are mechanisms that inactivate p16. 3 When inactivated, there is, in some cases, a loss of p16 protein expression in tumor cells as observed by immunohistochemistry. 6 In contrast, p16 is overexpressed in some tumors, commonly explained as secondary to Rb-inactivation. 4,7 The exact mechanism inducing p16 in Rb-inactivated tumors has not been elucidated, but both direct transcriptional effects and protein accumulation as a result of many population doublings have been proposed. 8 In tumors, many of the p16 mutations map to the cdk-binding regions ...

p16Ink4a overexpression in cancer: a tumor suppressor gene associated with senescence and high-grade tumors

“…These studies have shown that p16 Ink4a is expressed in the cytoplasm and the nucleus, depending on post-translational modifications or its capability to form a complex with other proteins (Nilsson and Landberg, 2006). p16 Ink4a interacts with several proteins located in the cytoplasm, such as a-b-g actin, a-b tubulin and CDK4/6 (Souza- Rodrigues et al, 2007).…”

Identification and Characterization of Bmi-1-responding Element within the Human p16 Promoter*

“…p16 and p19 are transcribed from the same gene, INK4a, which is tightly associated with the regulation of the cell cycle, senescence, and apoptosis (21,22). p16 inactivates Cdk, leading to repression of the retinoblastoma (Rb) gene and subsequent cell cycle arrest in the G1/S phase (23,24). p19 stabilizes p53 by antagonizing MDM2 and activating p53-dependent transcription and then arrests the cell cycle in G1 and G2/M phases, which in turn results in apoptosis (25,26).…”

Tumor Suppressor Gene p16/INK4A/CDKN2A and Its Role in Cell Cycle Exit, Differentiation, and Determination of Cell Fate

“…However, this speculation is not supported by the fact that p16 is expressed in both the nucleus and the cytoplasm in cell lines with wild-type p16 protein (Craig et al, 1998). Other studies have suggested that the cytoplasmic localization might represent a mechanism for p16 inactivation in various tumors (Evangelou et al, 2004;Nilsson & Landberg, 2006).…”

“…This phenomenon has been reported in normal cells where the protein was mainly found in the nucleus but not in the cytoplasm (Bartkova et al, 1996). However many tumor cell lines have been shown to harbor p16 in the cytoplasm as well as in the nucleus (Geradts et al, 2000;Nilsson & Landberg, 2006). Two major populations of p16 have been identified in subcellular fractions -one is unphosphorylated or basic in form and the other is phosphorylated or acidic in form and both are generally derived from post-translational modification.…”

“…It has been reported that the localization of the two forms of p16 in both cellular compartments mostly depends on cancer types. In breast cancer cell lines, both forms of p16 were observed in the cytoplasm while the phosphorylated form was predominant in the nucleus (Nilsson & Landberg, 2006). In addition, strong cytoplasmic expression of p16 was observed in many tumor cell lines including primary breast carcinoma associated with a malignant phenotype (Emig et al, 1998;Evangelou et al, 2004) suggesting that the protein might have specific roles for its cytoplasmic localization in certain malignancies.…”