Supplementary Experimental Procedures

YAP kinase screen (Kinasource)

Buffers: The kinase buffer for all non PKCs was 50mM Tris pH 7.5, 10mM 2-ME. The kinasebuffer for the conventional PKCs was 10mM HEPES pH 7.4, 10mM 2-ME, 0.5mM CaCl2, 0.25mg/ml phosphatidyl serine, 0.05mg/ml diacylglycerol, 0.01% Triton X-100. The kinasebuffer for the novel and atypical PKCs was 10mM HEPES pH 7.4, 10mM 2-ME, 0.5mM EGTA, 0.25mg/ml phosphatidyl serine, 0.05mg/ml diacylglycerol, 0.01% Triton X-100.

Substrates: 2.5µl Yap1 was used for the screen, 5µl were used for the initial rates and 10µl were used for the non radioactive samples.

Control substrates were: 0.4mg/ml Myelin Basic Protein for Erk2, SAPK2A, SAPK2B, SAPK3 and SAPK4; 0.1mg/ml GST-ATF2 for JNK1a1 and JNK2a2; 30µM Crosstide for AKT1/PKBa; 1YAP-S317A 00µM Chktide for Chk1 and Chk2; 50µM CK1tide for CK1d; 30µM 50µM AutoCaMtide for CaMKII; 0.1mg/ml GST-HSP27 for MAPKAP kinase 2 and PKD; 0.1mg/ml Histone H1 for PKC a, b and g; 0.1mg/ml PKCe substrate peptide for PKC e, q, z, and eta; 30µM Kemptide for PKA; 30µM longS6-peptide for p70S6K1 and p90RSK1; 30µM SPRKtide for SPRK and 30µM Srctide for Src.

Kinases: All kinases were sourced and used in their activated form. CaMKII was activated by autophosphorylation in the presence of Ca++/CaM and 200µM ATP.

Site-directed mutagenesis primer sequences

The following primers were used in the site directed mutagenesis of YAP at the five JNK phosphorylation sites from serine/threonine to alanine (A) residues and at two of the JNK phosphorylation sites, S317 and T362, from serine/threonine to aspartic acid (D).

YAP-T119A:5’-CTGCAGGAGCCCTGGCTCCACAGCATGTTCG-3’ and 5’-CGAACATGCTGTGGAGCCAGGGCTCCTGCAG-3’

YAP-S138A:5’-CAGTTGGGAGCTGTTGCTCCTGGGACACTGACC-3’ and 5’-GGTCAGTGTCCCAGGAGCAACAGCTCCCAACTG-3’

YAP-T154A:5’-CTCTGGCCCAGCAGCTGCACCCACAGCTCAGC-3’ and 5’-GCTGAGCTGTGGGTGCAGCTGCTGGGCCAGAG-3’

YAP-S317A:5’-CCAGTGTCTGCTCCCGGGATGTCTCAGG-3’ and 5’-CCTGAGACATCCCGGGAGCAGACACTGG-3’

YAP-T362A:5’-GCTACAGTGTCCCTCGAGCCCCAGATGACTTCC-3’ and 5’-GGAAGTCATCTGGGGCTCGAGGGACACTGTAGC-3’

YAP-S317D: 5'-gtgggactcaaaatccagtgtctgatcccgggatgtc-3' and 5'-gacatcccgggatcagacactggattttgagtcccac-3'

YAP-T362D:5'cagctacagtgtccctcgagacccagatgacttcctg-3' and 5'-caggaagtcatctgggtctcgagggacactgtagctg-3'

Supplementary Figure Legends

Supplementary Figure 1. ERK is not activated by anisomycin treatment

U2OS cells were treated with anisomycin or DMSO for 45 minutes. Cells were then harvested and analyzed by Western blot with indicated antibodies.

Supplementary Figure 2. YAP expression in HaCaT cells is protective from UV-induced apoptosis

(A) Examples of the Annexin V flow cytometry raw data clearly illustrate the difference in apoptosis levels between control shRNA and YAP shRNA HaCaT cell lines following UV irradiation. The lower left quadrant contains the live cells, the lower right contains the early apoptotic cells, the upper right quadrant contains the late apoptotic cells and the upper left contains the necrotic cells. (B) Lysates of HaCaT control and YAP shRNA cells used in the Annexin V assay were immunoblotted for expression of indicated proteins.

Supplementary Table 1: Results of the YAP kinase screen (Kinasource)

Table showing the amount of kinases and substrates used in the screen and the specific activity in U/mg of each kinase on YAP and control substrates.