Since CaMKII promotes neurite extension through the activation of the small GTPase RhoA in hippocampal neurons (Fink et al

Since CaMKII promotes neurite extension through the activation of the small GTPase RhoA in hippocampal neurons (Fink et al., 2003), we hypothesized that CaMKII might promote cytoskeletal rearrangements in TNBC cells in a RhoACdependent manner. cell culture setting, as well as an impairment in migration and invasion of TNBC cells. Finally, mice bearing xenografts of TNBC cells showed a significant delay in tumor growth when treated with small-molecule inhibitors of Pak and CaMKII. These data delineate a signaling pathway from Pak1 to CaMKII that is required for efficient proliferation, migration and invasion of mammary Salbutamol sulfate (Albuterol) epithelial cells, and suggest new therapeutic strategies in breast cancer. and kinase assays, we showed that Pak1 interacts with and phosphorylates CAMKII not only at T287, but also at T277. Moreover, we demonstrated that both, pharmacological inhibition of Pak1 activity or reduction of Pak1 expression through siRNA-mediated assays, significantly reduced the phosphorylation of CaMKII at T287. Conversely, the expression of a rapamycin-activatable Pak1 TGFB2 increased its phosphorylation levels. In addition, Pak1 and CaMKII are co-expressed in breast cancer cell lines and using a human breast cancer tissue microarray (TMA), we observed a significant correlation between the expression levels of these two kinases. Next, we showed that in a 3D cell culture setting, the combination of anti-Pak and anti-CaMKII agents has a synergistic inhibitory effect on cell proliferation, and induced apoptosis more potently in Her2 positive and TNBC cells, as well as an impairment in cell migration and invasion. Finally, we demonstrated that the combination of small molecule inhibitors targeting Pak1 and CaMKII significantly delayed the tumorigenesis of Salbutamol sulfate (Albuterol) TNBC cells in a xenograft setting. These data delineate a signaling pathway from Pak1 to CaMKII that is required for efficient proliferation, migration and invasion of mammary epithelial cells, and suggest new therapeutic avenues for the treatment of TNBC. Materials and Methods Antibodies and Reagents Antibodies used for western blot included anti Pak1, phospho-Pak1 (Ser199/204), CaMKII and phospho-CaMKII Thr287 pan-antibodies, myc-tag, cleaved caspase-3, PCNA and GAPDH from Cell Signaling Technology (Boston, MA. United States). Anti-GFP was from Santa Cruz Biotechnology (Dallas, TX. United States), and Anti-phospho-Threonine Antibody, clone 20H6.1, was from Sigma-Aldrich (St. Louis, MO. United States). DNA Constructs and siRNA The pCMV6M-Pak1 plasmid (Sells et al., 1997), was a gift from Jonathan Chernoff (Addgene plasmid 12209). The uniRapR-Pak1 mammalian expression vector (Dagliyan et al., 2017), was a gift from Klaus Hahn. The CaMKIIcDNA was amplified by PCR using the pDONR223-CAMK2G vector (Addgene plasmid 23409) as a template and cloned Salbutamol sulfate (Albuterol) into the HindIII/EcoRI restriction sites of pEGFP-C1 (Clonetech. Mountain View, CA. United States). The sequence encoding the regulatory domain (RD) of CaMKII(aminoacids 212C317) was amplified by PCR and cloned into the BamHI/EcoRI sites of pGEX-6P-2 (GE Healthcare. Braunschweig, Germany). The pGEX-6P-2-CaMKII 0.05; ** 0.01. Phospho-specific Protein Microarray Analysis The cancer signaling phospho-antibody arrays were purchased from Full Moon Biosystems, Inc. (Sunnyvale, CA. United States). Protein microarray analysis was performed following the manufacturers suggested protocol. Briefly, 100?g of cell lysates were diluted in 50?L of reaction mixture and labeled with biotin in 10?g/L N,N-dimethyformamide. The biotin-labeled proteins were diluted 1:20 in coupling solution before applying to the array for conjugation. To prepare the antibody microarray, it was blocked with blocking solution for 30?min at room temperature, rinsed with Milli-Q grade water, and dried with compressed nitrogen. Next, the array was incubated with the biotin-labeled proteins at 4C overnight, the arrays were washed three times with 60?ml of wash solution, and the conjugated-labeled proteins were detected using Cy3-streptavidin. Scanning, quantification, and data normalization were done using a G4900DA SureScan Microarray Scanner System and Feature Extraction v12.0 (Agilent Technologies). The extracted data were normalized and analyzed with the Subi platform (Subi Inc). Model of the Binding Complex of the Pak1 and Pak2 Kinase Domain and CaMKIIPeptide I and II To generate a model that could show the interaction of CaMKIIand its derived peptides I (sequence: LKHPWVCQRSTVASMMHRQET.