The CDK inhibitor p57Kip2 enhances the activity of the transcriptional coactivator FHL2

Abstract

The eukaryotic cell cycle is negatively regulated by cyclin-dependent kinase inhibitors (CKIs). p57 Kip2 is a member of the cip/Kip family of cKis and frequently inactivated by genomic mutations associated with human overgrowth disorders. There is increasing evidence for p57 to control cellular processes in addition to cell cycle and cDK regulation including transcription, apoptosis, migration or development. In order to obtain molecular insights to unknown functions of p57, we performed a protein interaction screen. We identified the transcription regulator four-and-a-half LIM-only protein 2 (FHL2) as a novel p57-binding protein. Co-immunoprecipitation and reporter gene assays were used to elucidate the physiological and functional relevance of p57/FHL2 interaction. We found in cancer cells that endogenous p57 and FHL2 are in a complex. We observed a substantial induction of established FHL2regulated gene promoters by p57 in reporter gene experiments and detected strong induction of the intrinsic transactivation activity of FHL2. Treatment of cells with histone deacetylase (HDAC) inhibitors and binding of exogenous FHL2 to HDACs indicated repression of FHL2 transcription activity byHDACs. In the presence of the HDAC inhibitor sodium butyrate activation of FHL2 by p57 is abrogated suggesting that p57 shares a common pathway with HDAC inhibitors. p57 competes with HDACs for FHL2 binding which might partly explain the mechanism of FHL2 activation by p57. These results suggest a novel function of p57 in transcription regulation.In order to gain more insight into novel functions of p57, we aimed to identify novel p57 binding partners. Therefore, we performed a yeast two-hybrid screen and obtained the protein FHL2 as a novel p57-interactor.FHL2 is a multifunctional LIM domain only protein which binds cellular proteins via its LIM domains and thereby regulates various cellular processes 18 . Although FHL2 does not directly bind to DNA, it modulates the activity of several transcription factors 19,20 . FHL2 was first described to bind to the hormone-activated androgen receptor (AR) which increases the activity of AR-dependent reporter genes 21 . FHL2 is expressed in the cytoplasm and the nucleus. Interestingly, in several cancer types high levels of nuclear FHL2 correlate with disease progression towards a malignant state. This indicates that FHL2 dependent transcription contributes to cancer development and progression 22,23 .Here we report that p57 strongly activates FHL2 transactivation function and induces the activity of known FHL2-regulated promoters. We provide experimental evidence supporting the hypothesis that FHL2 is repressed by HDACs and p57 relieves this repression by competing with HDACs for FHL2-binding. FHL2 and p57 might regulate transcription as components of chromatin remodeling complexes. Materials and Methodsplasmids and oligonucleotide sequences. Detailed descriptions of novel plasmid constructs, including cloning strategies and sequences of oligonucleotides used are presented in Supplementary inf...

Stimulation of c-Jun/AP-1-Activity by the Cell Cycle Inhibitor p57Kip2

“…Expression vectors pDEST-3xHA-p57, pDEST-3xFLAG-p57, pDEST-3xHA-p57-CK-, pDEST-3xHA-p57-Nt, pDEST-3xHA-p57-Nt-NLS1, pDEST-3xHA-p57-Ct, pDEST-3xHA-p27, and pDEST-3xHA-p21 have been already described ( Kullmann et al, 2020 ). In pDEST-3xHA-p57-CK- the wildtype human p57 sequence codes for a mutant protein (R31A_L33A_W61A_F65A) not binding to Cyclins and CDKs.…”

“…Either by direct binding to MyoD or by inhibiting cyclin E/CDK2 dependent destabilization of MyoD, p57 was able to promote myogenesis in cell culture models ( Reynaud et al, 1999 , 2000 ). Recently, we found that p57 is able to activate gene promoters by interacting with the LIM domain-only protein FHL2 ( Kullmann et al, 2020 ). FHL2 coactivates AP-1 complexes, where the proto-oncoproteins c-Jun and c-Fos form the most active AP-1 heterodimeric complexes ( Morlon and Sassone-Corsi, 2003 ).…”

“…FHL2 coactivates AP-1 complexes, where the proto-oncoproteins c-Jun and c-Fos form the most active AP-1 heterodimeric complexes ( Morlon and Sassone-Corsi, 2003 ). p57 binds to FHL2 and increases its intrinsic transactivation, probably by competing with inhibitory histone deacetylases (HDACs) for FHL2 binding ( Kullmann et al, 2020 ). AP-1 is composed of homo- or heterodimeric complexes consisting of transcription factors belonging to the basic-leucine zipper (bZIP) family ( Hess et al, 2004 ).…”

Functional Versatility of the CDK Inhibitor p57Kip2

“…Finally, it was shown recently that p57 may also positively regulate transcription ( Kullmann et al, 2020 ). p57 binds to and activates FHL2 (four-and-a-half LIM only protein 2), a multifunctional LIM domain only protein, which binds to and modulates the activity of several transcription factors, such as AP-1, β-catenin or the androgen receptor, by acting as a coactivator ( Muller et al, 2000 ; Tran et al, 2016 ).…”

“…p57 binds to and activates FHL2 (four-and-a-half LIM only protein 2), a multifunctional LIM domain only protein, which binds to and modulates the activity of several transcription factors, such as AP-1, β-catenin or the androgen receptor, by acting as a coactivator ( Muller et al, 2000 ; Tran et al, 2016 ). FHL2 is inhibited by HDACs, and by binding to FHL2, p57 competes with HDAC1 and HDAC3 for FHL2 binding and prevents its inhibition ( Kullmann et al, 2020 ).…”