Catalytic activity of nuclear PLC-β 1 is required for its signalling function during C2C12 differentiation ☆ Giulia Ramazzotti a, 1 , Irene Faenza a, 1 , Gian Carlo Gaboardi a , Manuela Piazzi a,b , Alberto Bavelloni a,b , Roberta Fiume a , Lucia Manzoli a , Alberto M. Martelli a,c , Lucio Cocco a, ⁎ a Cellular Signalling Laboratory, Department of Human Anatomical Sciences, University of Bologna, 40126 Bologna, Italy b Laboratory of Cell Biology and Electron Microscopy, Istituti Ortopedici Rizzoli, 40136 Bologna Italy c IGM-CNR, Sezione di Bologna c/o I.O.R., Bologna, Italy abstract article info Article history: Received 30 June 2008 Received in revised form 15 July 2008 Accepted 16 July 2008 Available online xxxx Keywords: Phospholipase C-β1 Nuclear inositide signalling Cyclin D3 Myogenic differentiation Here we report that PLC-β 1 catalytic activity plays a role in the increase of cyclin D3 levels and induces the differentiation of C2C12 skeletal muscle cells. PLC-β 1 mutational analysis revealed the importance of His 331 and His 378 for the catalysis. The expression of PLC-β 1 and cyclin D3 proteins is highly induced during the process of skeletal myoblast differentiation. We have previously shown that PLC-β 1 activates cyclin D3 promoter during the differentiation of myoblasts to myotubes, indicating that PLC-β 1 is a crucial regulator of the mouse cyclin D3 gene. We show that after insulin treatment cyclin D3 mRNA levels are lower in cells overexpressing the PLC-β 1 catalytically inactive form in comparison to wild type cells. We describe a novel signalling pathway elicited by PLC-β 1 that modulates AP-1 activity. Gel mobility shift assay and supershift performed with specific antibodies indicate that the c-jun binding site is located in a cyclin D3 promoter region specifically regulated by PLC-β 1 and that c-Jun binding activity is significantly increased by insulin and PLC-β 1 overexpression. Mutation of AP-1 site decreased the basal cyclin D3 promoter activity and eliminated its induction by insulin and PLC-β 1 . These results hint at the fact that PLC-β 1 catalytic activity signals a c-jun/ AP-1 target gene, i.e. cyclin D3, during myogenic differentiation. © 2008 Elsevier Inc. All rights reserved. 1. Introduction It is now evident that phosphoinositide signalling circuitry, involving phosphatidylinositol 4,5-bisphosphate (PIP 2 ), its synthesiz- ing enzymes, i.e. type I phosphatidylinositol 4-phosphate kinase α [1], and its metabolizing enzymes, like PLC-β 1 , is present at the plasma membrane and in the nucleus [2,3]. The presence of PLC-β 1 and its substrates at both sites suggests that its localisation and catalytic activity are finely regulated and that the distinct pools of inositol 1,4,5- trisphosphate (IP 3 ) released in the nucleus and cytoplasm have different fates and physiological roles. PLC-β 1 is a key player in the regulation of nuclear inositol lipid signalling and of a wide range of cellular functions, such as proliferation, survival, differentiation and metabolism [4]. PLC-β 1 belongs to the family of PLCs which are soluble multi-domain proteins ranging in molecular masses from 85 to 150 kDa. PLCs are subdivided in six major families (beta, gamma, delta, epsilon, zeta and eta), which comprise at least 13 isoforms [5]. These isozymes show different structural organization as well as regulation and tissue distribution. In general beta, gamma and delta isoforms contain a N-terminal pleckstrin homology (PH) domain, followed by an EF-hand domain, a catalytic domain (X/Y domain) and a C2 domain. PLC-βs differ from other PLCs because of a 400 residue- long C-terminal extension, located downstream the C2 domain, which is important for the nuclear localisation and for G protein activation. The comparison of different PLCs' amino acid sequences indicates that the catalytic domain represents the region with higher sequence similarity among the different families. In particular several residues belonging to the X and Y domains are conserved or replaced conservatively in all eukaryotic PLCs [6]. Moreover in a structural study on rat PLC-δ1, the authors demonstrated that two of these amino acids, namely His-311 and His-356, play a determinant role in the acid/ base catalytic process [7]. Up to now our group has demonstrated that PLC-β 1 nuclear loca- lisation is crucial for its function. Presently, our aim is to characterize better the mechanism by which PLC-β 1 induces its signalling network. In the past years, PLC-β 1 has been implicated in the control of distinct differentiation systems. In particular, cyclin D3 activation represents a common element of PLC-β 1 activity in the differentiation process of both erythroleukemia MEL cells [8] and skeletal muscle C2C12 cells [9,10]. The C2C12 myogenic cell line has been widely used to study the involvement of PLC-β 1 in muscle cell differentiation. Cellular Signalling xxx (2008) xxx–xxx ☆ This work was supported by Italian MIUR-FIRB, MIUR-COFIN and CARISBO Foundation. ⁎ Corresponding author. Cellular Signalling Laboratory, Department of Human Anatomical Sciences, University of Bologna, Via Irnerio 48, 40126 Bologna, Italy. E-mail address: lucio.cocco@unibo.it (L. Cocco). 1 Contributed equally to this work. CLS-06707; No of Pages 9 0898-6568/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2008.07.009 Contents lists available at ScienceDirect Cellular Signalling journal homepage: www.elsevier.com/locate/cellsig ARTICLE IN PRESS Please cite this article as: G. Ramazzotti, et al., Cell. Signal. (2008), doi:10.1016/j.cellsig.2008.07.009