br Materials and methods br Results br Discussion ERK signal

Materials and methods
Results
Discussion ERK5 signaling and its regulation has gained focus in recent years specially in the subject of cardiovascular biology, where this kinase plays a key role in angiogenesis and maintenance of blood vessel integrity [18,20,34,35]. The signaling pathways induced by active ERK5 also regulate other critical cellular processes including migration and survival [16,21,36]. ERK5 has been shown to promote PDGF-induced anti-apoptotic signaling, where it activates AKT in a pathway involving SRC and the phosphatase SHP2 [36]. In migrating cells, ERK5 can trigger Pancuronium dibromide remodeling and enhances invasiveness of breast cancer cells [37,38]. However, comprehensive links between ERK5 and the elaborate cellular machinery, which is involved in cytoskeleton regulation have not been established so far. Similarly, integrin signaling has been associated to the ERK5 pathway through FAK, but the role of ERK5 in adhesion-dependent downstream processes is less clear [15]. Here we aimed to understand the role of ERK5 in pro-survival signaling, induced by cell-matrix interactions primarily mediated through integrins. We used CRISPR/Cas9 technique to knock-out ERK5 in different cancer cell lines and then chose a widely used breast cancer line MDA-MB231 to study the consequences of ERK5 depletion on adhesion-dependent cell survival. Two out of three sgRNAs designed by us provided efficient ERK5 knock-out when expressed with Cas9 in a single vector (U6-gRNA-spCas9-2A-GFP) format (Fig. 1A). This indicates that an initial screening approach should be adopted by using 3 to 4 sgRNAs designed against a desired target, to identify the guide that offers best editing with low cell toxicity. Integrins β1 and β3 subunits form complexes with different alpha subunits such as αV, α1, α7 etc. and act as receptors for various extracellular matrix ligands [39]. These form integral components of the cellular adhesosome, which regulates among many other processes, survival and anoikis of adherent cells. Cell adhesion in-turn also triggers signaling cascades and adhesion mediated pro-survival signaling is one of the many downstream attributes of this process [33,39,40]. FAK is the primary cytosolic kinase that acts both as a scaffold and an enzyme in adhesion-induced signaling and promotes ERK1/2 and ERK5 phosphorylation [41]. FAK is auto-phosphorylated at Y397 in response to integrin engagement or growth factor stimulation and this site is used as a read-out of FAK activation. But differential FAK phosphorylation at additional sites is also known to play an important role in FAK-mediated signaling and its regulation [27,42,43]. Recently, ERK5 was demonstrated as the kinase directly responsible for FAK S910 phosphorylation in response to PMA stimulation [27,28]. Here we showed that in addition to Y397, cell-adhesion can also induce ERK5-dependent FAK S910 phosphorylation (Fig. 2A). However, the mechanistic details and cellular functions of FAK phosphorylated at S910 remain largely unclear and require more investigation. Though FAK is the main modulator of integrin signaling, there is evidence that in the absence of FAK, a related kinase PYK2 can compensate some of its functions [44]. Like FAK, PYK2 activation is also triggered by several up-stream receptors, including integrins and growth factor receptors [45]. In human breast cancers, PYK2 is thought to perform a pivotal role by serving as a node where growth factor and cytokine signaling converges [46]. Unlike FAK S910, direct phosphorylation of PYK2 by ERK5 has not been observed. However, in our work FAK silencing in ERK5 knock-out MDA-MB231 cells, on its own, did not result in a significant impact on cell adhesion (Fig. 3B). But simultaneous inhibition of FAK and PYK2 in ERK5 KO cells reduced FN adhesion and proved detrimental for adhesion-induced cell survival (Fig. 3, Fig. 4B). Still, it remains unknown how PYK2 and ERK5 interact and affect each other. In a recent report, it was found that ERK5 inhibition using a chemical inhibitor was enough to increase apoptosis in acute myeloid leukemia cell lines [47]. Our data supports this finding as depletion of ERK5, by itself, triggered the activation of apoptotic signaling in adherent MDA-MB231 cells as suggested by the observation of cleaved PARP and caspase 9 bands in DMSO treated ERK5 KO cells (Fig. 4C). However, apoptosis in these cells was enhanced when FAK and PYK2 were prevented from their functions by the dual inhibitor PF 431396.