HEK293T/17 cells transfected with FLAG-empty vector (EV), FLAG-wild-type cortactin (WT) or the indicated FLAG-cortactin mutants. of cortactin T24 by CK2 impairs the ability of cortactin to bind Arp2/3 and activate actin nucleation. Decreased invadopodia activity is observed in HNSCC cells with expression of CK2 phosphorylation-null cortactin mutants, shRNA-mediated CK2 knockdown, and with the CK2 inhibitor Silmitasertib. Silmitasertib inhibits HNSCC collective invasion in tumor spheroids and orthotopic tongue tumors in mice. Collectively these data suggest that CK2-mediated cortactin phosphorylation at T24 is critical in regulating cortactin binding to Arp2/3 complex and pro-invasive activity, identifying a potential targetable mechanism for impairing HNSCC invasion. Implications: This study identifies a new signaling pathway that contributes to enhancing cancer cell invasion. kinase assays were performed as described (30). Briefly, 0.25, 0.5, or 1 g of purified GST-WT or T24A cortactin NTA fusion proteins were incubated with 8 ng CK2 (#14-445, Millipore) and 10 Ci 32P-ATP (#NEG002A500UC, PerkinElmer) at 30C for 10 minutes. Reactions were terminated with hot SDS sample loading buffer. Proteins were visualized by autoradiography. Purified N-WASp GST-VCA (0.5 g) and GST Gabapentin Hydrochloride (1 g) were used as respective positive and negative controls. cortactin phosphorylation binding assay Purified WT or T24A cortactin proteins (2.5 g) were bound to 4F11-conjugated protein G magnetic beads (#10003D, Life Technologies). Immune complexes were incubated in the presence or absence of activated CK2 (75 ng; #V4482, Promega) and ATP (500 nmoles, #BP413-25, Fisher Scientific) at 30C for 15 minutes. Reactions were washed twice with 10 mM Tris pH 7.4, 150 mM NaCl, 0.5 mM EDTA. Complexes were washed once with 10 mM Tris pH 7.4, 10 mM EDTA and incubated with 50 ng Arp2/3 Rabbit polyclonal to AMPKalpha.AMPKA1 a protein kinase of the CAMKL family that plays a central role in regulating cellular and organismal energy balance in response to the balance between AMP/ATP, and intracellular Ca(2+) levels. complex (#RP01-A, Cytoskeleton) at 4C for 30 minutes. Following incubation, binding complexes were washed once with 10 mM Tris Buffer pH 7.4 with 25 mM NaCl, 10 mM EDTA, 1% NP-40, then boiled and Western blotted with antibodies against cortactin and Arp3. Actin polymerization assay Actin polymerization experiments were conducted as described previously (31). Reactions contained 2 M actin (10% pyrene-labeled), 75 nM Arp2/3 complex, 100 nM cortactin or 50 nM GST-VCA (#VCG03, Cytoskeleton), and/or varying amounts of CK2 (#14-445, Millipore) as indicated. For reactions with CK2, GST-VCA or Gabapentin Hydrochloride cortactin mutants were preincubated with CK2 and 500 nmoles ATP for 15 minutes at room temperature prior to addition to the actin polymerization reaction. PDX-derived cell lines Patient-derived xenograft (PDX) tumors and cell lines were established as described (32). WVUSCC-AR2 and WVUSCC-AR5 were derived from surgical specimens of alveolar ridge HNSCC in compliance with West Virginia University Institutional Review Board approved protocol #1310105737A033. PDXs were developed in compliance with West Virginia University Institutional Animal Care and Use Committee approved protocol #15-0302.6 by placing approximately 1 mm tumor fragments into subcutaneous pockets in the flanks of anesthetized 8-10 week old NOD/SCID- (NSG) mice. Tumor fragments were overlayed with Matrigel (354234, Corning) and incisions were closed using wound clips. Mice were weighed and monitored for tumor growth on a weekly basis. PDX tumors were passed into new NSG mice and/or used to generate cell lines once tumors reached ~1 cm in greatest Gabapentin Hydrochloride dimension. For cell line derivation, PDX tumors were minced and digested in DMEM supplemented with 20% FBS and 1 mg/mL collagenase IV (17104019, Gibco). Digested tissues were plated onto NIH3T3 fibroblasts senesced with 4 g/mL mitomycin C (BP2531, Fisher) and cultured in DMEM:F12 1:1 supplemented with 10% FBS, 400 ng/mL hydrocortisone (H0888, Sigma), 50 g/mL gentamycin (15750060, Gibco), 5 M ROCK inhibitor (S1049, Selleckchem), 0.5 ng/mL recombinant human epidermal Gabapentin Hydrochloride growth factor (EGF) (PHG0311, Gibco), and 10 ng/mL cholera toxin.