Cell cycle control by optogenetically regulated cell cycle inhibitor protein p21

The cell cycle is divided in four phases, the G1 phase for growth in cell size and increased protein biosynthesis, the S phase for the synthesis and replication of DNA, and the G2 phase for preparing the cell for the M phase, the phase of cell division. Cell cycle inhibitors control progression through the cell cycle. The cell cycle inhibitor p21 arrests cells in the G1 phase correlating with a prolonged protein production phase. This effect could be used to increase the production of biotherapeutic proteins. Here, we applied an optogenetic approach to control the function of p21. Optogenetics is an emerging field within synthetic biology and based on genetically encoded light-sensitive elements derived from plants, fungi or bacteria. Optogenetic tools can be used to control biological functions such as signaling pathways, metabolic pathways or gene expression via light with less side effects than when using chemical inducers. In this study, we designed and applied light switches to control the subcellular localization and thereby the function of p21via light. The stimulation of light-regulated p21 increased the number of cells arrested in the G1 phase correlating with the increased expression of a reporter protein. Implementation of this system could be used to optimize the production of biotherapeutic protein.German Research Foundation (DFG)Germany’s Excellence Strateg

CNK1 and other scaffolds for Akt/FoxO signaling

AbstractFoxO transcription factors mediate anti-proliferative and pro-apoptotic signals and act as tumor suppressors in cancer. Posttranslational modifications including phosphorylation and acetylation regulate FoxO activity by a cytoplasmic–nuclear shuttle mechanism. Scaffold proteins coordinating signaling pathways in time and space play a critical role in this process. CNK1 acts as a scaffold protein in several signaling pathways controlling the function of FoxO proteins. An understanding of CNK1 and other scaffolds in the FoxO signaling network will provide insights how to release the tumor suppressor function of FoxO as a possibility to block oncogenic pathways. This article is part of a Special Issue entitled: P13K-AKT-FoxO axis in cancer and aging

CNK1 Promotes Invasion of Cancer Cells through NF-κB–Dependent Signaling

Abstract Hallmarks of cancer cells are uncontrolled proliferation, evasion of apoptosis, angiogenesis, cell invasion, and metastasis, which are driven by oncogenic activation of signaling pathways. Herein, we identify the scaffold protein CNK1 as a mediator of oncogenic signaling that promotes invasion in human breast cancer and cervical cancer cells. Downregulation of CNK1 diminishes the invasiveness of cancer cells and correlates with reduced expression of matrix metalloproteinase 9 (MMP-9) and membrane-type 1 MMP (MT1-MMP). Ectopic expression of CNK1 elevates MT1-MMP promoter activity in a NF-κB–dependent manner. Moreover, CNK1 cooperates with the NF-κB pathway, but not with the extracellular signal–regulated protein kinase pathway, to promote cell invasion. Mechanistically, CNK1 regulates the alternative branch of the NF-κB pathway because knockdown of CNK1 interferes with processing of NF-κB2 p100 to p52 and its localization to the nucleus. In agreement with this, the invasion of CNK1-depleted cells is less sensitive to RelB downregulation compared with the invasion of control cells. Moreover, CNK1-dependent MT1-MMP promoter activation is blocked by RelB siRNA. Thus, CNK1 is an essential mediator of an oncogenic pathway involved in invasion of breast and cervical cancer cells and is therefore a putative target for cancer therapy. Mol Cancer Res; 8(3); 395–406</jats:p

Specific association of Mil/Raf proteins with a 34 kDa phosphoprotein

Mil/Raf protein kinases are intermediates in signaling pathways leading to differentiation, mitogenesis and cellular transformation. To gain insight into the activity of Mil/Raf kinases at the molecular level we aimed to identify proteins specifically interacting with Mil/Raf proteins. A phosphoprotein of 34 kDa (pp34) was found to be associated with c-Raf as well as with viral and activated forms of Mil/Raf proteins in exponentially growing interphase cells. pp34 association was not detectable in mitotic cells. Serum stimulation or coexpression of activated Ras led to decreased electrophoretic mobility of pp34 complexed to Mil/Raf proteins while serum starvation rendered pp34 undetectable. Moreover, the association with pp34 became undetectable in parallel with the onset of morphological cellular transformation caused by overexpression of a constitutively activated mutant of c-Raf in an inducible expression system. Thus, the association of Mil/Raf proteins with pp34 is altered in the course of cell cycle progression, serum stimulation and cellular transformation. These events represent hallmarks of cellular Mil/Raf functions, rendering pp34 a candidate protein involved in Mil/Raf function