Journal: Cancers

Article Title: Rac1 GTPase Regulates the βTrCP-Mediated Proteolysis of YAP Independently of the LATS1/2 Kinases

doi: 10.3390/cancers16213605

Figure Lengend Snippet: YAP degradation after Rac1 inhibition requires the SCF βTrCP E3 ubiquitin ligase. ( A ) Time course of YAP decline after Rac1 inhibition. HPAF/CD18 cells were harvested at the indicated time point after the addition of EHT-1864 (50 μM). Levels of YAP and S127-phosphorylated YAP were quantified by immunoblotting. GAPDH was used as an internal standard. ( B ) MG132 blocks the degradation of YAP elicited by the inhibition of Rac1. After 12 h of exposure to EHT-1864 (50 μM; +) or vehicle control (−), HPAF/CD18 cells were exposed or not to MG132 (20 μg/mL) for 4 h prior to harvesting. ( C ) Schematic description of YAP structure showing the position and sequence of critical degrons and phosphorylation sites. Upper drawing shows the structure of YAP, including its heterodimerization domain (TEAD), WD40 domains (WW), transactivation domain (TAD), putative degrons (βTrCP1/2, FBXW7), and LATS1/2 phosphorylation sites (S61, S109, S127, S128, S131, S163, S164, and S381). Lower left panel: Sequence of the putative FBXW7 phosphodegron highlighting its consensus and its potentially required phosphoserine group (S351). Changes introduced by the S351A/P352A mutation are also shown. Lower right panel: Sequence of the βTrCP degron of YAP highlighting its consensus and its required CK1 (S384, S387) and LATS1/2 (S381) phosphorylation sites. Changes introduced by the D383A/S384A mutation are also shown. ( D ) Detection of the Flag-YAP proteins in retrovirally infected Panc1 cells. Panc1 cells infected with pLXSH viruses carrying no insert (Empty), Flag-tagged YAP (WT), or its various mutant versions (5SA, D383A/S384A, S351A/P352A) were analyzed for the presence of Flag-YAP. GAPDH was used as an internal control. ( E ) The βTrCP degron is needed for YAP degradation after Rac1 inhibition, but not the S381 LATS1/2 phosphorylation site. In duplicate, Panc1 cells expressing the different mutants of Flag-YAP were exposed to EHT-1864 (50 μM). Sixteen hours later, Flag-tagged proteins were quantified using western blotting. Again, GAPDH was used as an internal control. ( F ) The siRNA-mediated knockdown of Skp1 blocks YAP degradation after Rac1 inhibition. Panc1 cells were transfected with skp1 siRNA or with a non-targeting siRNA. Forty-eight hours later, transfected cells were treated with EHT-1864 (50 μM; +) or vehicle control (−) for 16 h prior to western blot analysis. ( G ) The siRNA-mediated knockdown of the βTrCP1/2 proteins blocks YAP degradation after Rac1 inhibition. Panc1 cells were transfected with a non-targeting siRNA or with siRNA against βTrCP1, βTrCP2, or both proteins. Forty-eight hours later, transfected cells were treated with EHT-1864 (50 μM; +) or vehicle control (−) for 16 h prior to western blot analysis. GAPDH was again used as an internal standard. For all panels, densitometry readings for the indicated intensity ratios (pYAP/GAPDH, YAP/GAPDH, Skp1/GAPDH, and FLAG/GAPDH) are shown below the lanes of each western blot.

Article Snippet: PCR products encoding Flag-tagged YAP and its 5SA mutant were respectively amplified from vectors p2xFlag CMV2-YAP2 (a gift from Marius Sudol’s lab; purchased from Addgene cat# 19045) and pCMV2-flag YAP2 5SA (a gift from Kunliang Guan’s lab; purchased from Addgene cat# 27371) using the same forward (5′-GTACGC GTCGAC AGTGAACCGTCAGAATTGATCTA-3′; Sal1 site underlined) and reverse (5′-CATGGA AGATCT CTATAACCATGTAAGAAAGCTT-3′; Bgl2 site underlined) primers.

Techniques: Inhibition, Ubiquitin Proteomics, Western Blot, Control, Sequencing, Phospho-proteomics, Mutagenesis, Infection, Expressing, Knockdown, Transfection

Journal: Cancers

Article Title: Rac1 GTPase Regulates the βTrCP-Mediated Proteolysis of YAP Independently of the LATS1/2 Kinases

doi: 10.3390/cancers16213605

Figure Lengend Snippet: YAP degradation after Rac1 inhibition is LATS1/2-independent but requires CK1. ( A ) The silencing of LATS1/2 fails to prevent YAP degradation after Rac1 inhibition. Panc1 cells were transfected with a non-targeting siRNA or with siRNA against LATS1, LATS2, or both proteins. Forty-eight hours later, transfected cells were treated with EHT-1864 (50 μM; +) or the vehicle control (−) for 16 h prior to western blot analysis. GAPDH was again used as an internal standard. ( B ) Detection of LATS1 and LATS2 in the LATS1/2-proficient and -deficient HeLa cells. Cells were probed with the indicated antibodies. ( C , D ) LATS1/2 are not needed for YAP degradation after Rac1 inhibition. LATS1/2-proficient and -deficient cells HeLa cells were exposed to 50 μM EHT-1864 ( C ) or the DMSO vehicle ( D ) for 16 h, after which YAP levels were measured. ( E , F ) Panc1 cells expressing the Flag-YAP protein ( E ) or its 5SA mutant ( F ) were exposed or not to CK1 inhibitor IC-261 (10 μM), after which YAP levels were measured. GAPDH was used as an internal standard. For all panels, densitometry readings for the indicated intensity ratios (pYAP/GAPDH, YAP/GAPDH, LATS1/GAPDH, and FLAG/GAPDH) are shown below the lanes of each western blot.

Article Snippet: PCR products encoding Flag-tagged YAP and its 5SA mutant were respectively amplified from vectors p2xFlag CMV2-YAP2 (a gift from Marius Sudol’s lab; purchased from Addgene cat# 19045) and pCMV2-flag YAP2 5SA (a gift from Kunliang Guan’s lab; purchased from Addgene cat# 27371) using the same forward (5′-GTACGC GTCGAC AGTGAACCGTCAGAATTGATCTA-3′; Sal1 site underlined) and reverse (5′-CATGGA AGATCT CTATAACCATGTAAGAAAGCTT-3′; Bgl2 site underlined) primers.

Techniques: Inhibition, Transfection, Control, Western Blot, Expressing, Mutagenesis