Tyrosine Phosphorylation of Caspase-8 Abrogates Its Apoptotic Activity and Promotes Activation of c-Src.

Src family tyrosine kinases (SFKs) phosphorylate caspase-8A at tyrosine (Y) 397 resulting in suppression of apoptosis. In addition, the phosphorylation of caspase-8A at other sites including Y465 has been implicated in the regulation of caspase-8 activity. However, the functional consequences of these modifications on caspase-8 processing/activity have not been elucidated. Moreover, various Src substrates are known to act as potent Src regulators, but no such role has been explored for caspase-8. We asked whether the newly identified caspase-8 phosphorylation sites might regulate caspase-8 activation and conversely, whether caspase-8 phosphorylation might affect Src activity. Here we show that Src phosphorylates caspase-8A at multiple tyrosine sites; of these, we have focused on Y397 within the linker region and Y465 within the p12 subunit of caspase-8A. We show that phosphomimetic mutation of caspase-8A at Y465 prevents its cleavage and the subsequent activation of caspase-3 and suppresses apoptosis. Furthermore, simultaneous phosphomimetic mutation of caspase-8A at Y397 and Y465 promotes the phosphorylation of c-Src at Y416 and increases c-Src activity. Finally, we demonstrate that caspase-8 activity prevents its own tyrosine phosphorylation by Src. Together these data reveal that dual phosphorylation converts caspase-8 from a pro-apoptotic to a pro-survival mediator. Specifically, tyrosine phosphorylation by Src renders caspase-8 uncleavable and thereby inactive, and at the same time converts it to a Src activator. This novel dynamic interplay between Src and caspase-8 likely acts as a potent signal-integrating switch directing the cell towards apoptosis or survival

Positive feedback loop of caspase-8A induced Src activation.

<p>Phosphorylation of caspase-8A by Src at Y465 prevents its cleavage, and so prevents the induction of apoptosis. The presence of inactive caspase-8A (phosphorylated at Y465) allows the phosphorylation of Y397. When Y397 is phosphorylated in the presence of Y465 phosphorylation, caspase-8A then becomes a Src activator, by binding to the SH2 domain of Src via its pY₃₉₇LEM peptide, further activating Src by promoting Y416 phosphorylation of Src.</p

Y465 phosphomimetic modification of caspase-8A abolishes its ability to activate caspase-3 and to induce apoptosis.

<p>WT GFP-caspase-8A mutants were transfected into HEK293 cells. A) Cells were fixed and immunostained with anti-cleaved caspase-3 antibody. Green represents GFP-caspase-8A expressing cells; orange represents active caspase-3 in GFP-caspase-8A expressing cells (white arrow). B) The percentage of active caspase-3 positive cells is displayed. N = 3, * <i>p</i> < 0.05. C) Cell morphology of transfected cells was examined using live fluorescence microscopy. Healthy cells are identified with orange arrows whereas apoptotic cells characterized by cell rounding or apoptotic bodies are identified with white arrows. D) Transfected cells were permeabilized and stained with propidium iodide to qualified the percentage of cells expressing hypodiploid DNA representing DNA fragmentation seen in apoptosis. N = 3, <i>p</i> < 0.05.</p

Inhibition of caspase-8 activity enhances Src-dependent tyrosine phosphorylation of caspase-8.

<p>A) HEK293 cells were transfected with GFP-caspase-8A (WT or C377S inactive mutant) with or without Y527F Src for 24 hours. Whole cell lysates were subjected to Western blot analysis with anti-phospho-tyrosine antibody and anti-GFP antibody. B) HEK293 cells were transfected with GFP-caspase-8A (WT or C377S inactive mutant) with Y527F Src for 24 hours. GFP-caspase-8A was immunoprecipitated with anti-GFP antibody and precipitates were probed with anti-phospho-tyrosine antibody and anti-GFP antibody. C) HEK293 cells were transfected with WT GFP-caspase-8A and Y527F Src for 9 hours followed by treatment with DMSO vehicle control or caspase-8 inhibitor (20 μM) for 15 hours. Whole cell lysates were subjected to Western blot analysis with anti-phospho-tyrosine antibody and anti-GFP antibody.</p

List of Primers Used for Point Mutations of GFP-Caspase-8 Constructs.

<p>List of Primers Used for Point Mutations of GFP-Caspase-8 Constructs.</p

Y465 phosphomimetic modification of caspase-8A promotes Src activation and activity.

<p>HEK293 cells were co-transfected with empty GFP plasmid or GFP-tagged caspase-8A (WT and various mutants) and Y527F Src for 24 hours. A) Whole cell lysates were subjected to Western blot analysis with antibodies to pY416 Src and Src. Src Y416 phosphorylation was normalized to total Src. N = 12, <i>p</i> < 0.0001. B) HEK293 cells were co-transfected with GFP-caspase-8A with or without Y527F Src for 24 hours. Y527F Src was immunoprecipitated using an anti-avian Src antibody and probed with anti-avian Src and anti-pY416 Src antibody to demonstrate specificity of anti-avian Src antibody. C) HEK293 cells were co-transfected with empty GFP plasmid or GFP-caspase-8A (WT and various mutants) with Y527F Src for 24 hours; Y527F Src was immunoprecipitated using an anti-avian Src antibody then probed with anti-avian Src and anti-pY416 Src antibodies. Y416 phosphorylation signal of avian Src was normalized to total expressed avian Src level. N = 3, <i>p</i> = 0.02. D) Whole cell lysates were subjected to Western blot analysis with anti-phospho-tyrosine antibody to show global protein tyrosine phosphorylation. The same blot was also probed with anti-GFP antibody to look at the expression of transfected GFP-caspase-8A; anti-avian Src antibody to look at the expression of transfected avian Src; and anti-beta actin antibody to look at the expression of beta actin as a loading control. E) Whole cell lysates were subjected to Western blot analysis to evaluate Erk1/2 phosphorylation. Phosphorylation of Erk1/2 was normalized to total expressed Erk1/2 level. N = 8, <i>p</i> < 0.001. F-H) HEK293 (F), A549 (G) and CHO (H) cells were co-transfected with empty GFP plasmid or GFP-tagged caspase-8B (WT and various mutants) with Y527F Src for 24 hours; Y527F Src was immunoprecipated using anti-avian Src antibody. Then precipitates were immunoblotted with antibodies to pY416 Src and avian Src.</p

Tyrosine phosphorylation of caspase-8A in multiple sites follows Src expression.

<p>A) Caspase-8A structure and SFK tyrosine phosphorylation sites predicted by GPS2.1. B) HEK293 cells were co-transfected with inactive C377S mutant of GFP-caspase-8A and Y527F Src for 24 hours then lysates immunoprecipitated using an anti-GFP antibody. GFP-caspase-8A IP was subjected to SDS-PAGE, and the appropriate band (80 kDa) was excised and sent for LC-MS/MS analysis. C) Mutagenesis was performed at Y397/Y380 and Y465/Y448 sites to generate phosphomimetic (tyrosine to glutamic acid) and non-phosphorylatable (tyrosine to phenylalanine) caspase-8A/B mutants.</p

Y465 phosphomimetic mutation inhibits caspase-8A cleavage.

<p>A) Upon activation, caspase-8 is autocatalytically cleaved first after D391, and then after D233. Using an anti-GFP antibody that binds to the N-terminus of the molecule, a 68-kDa fragment is visualized when GFP-caspase-8A is cleaved after D391, whereas a 50-kDa fragment is visualized when GFP-caspase-8A is cleaved after D233. B) Using an anti-caspase-8 antibody that binds to the C-terminus of the molecule, a 30-kDa fragment is visualized when GFP-caspase-8A is cleaved after D233, whereas a 12-kDa fragment is visualized when GFP-caspase-8A is cleaved after D391. A 55-kDa band represents endogenous caspase-8. C) HEK293 cells were transfected with GFP-caspase-8A WT or mutants for 24 hours. Whole cell lysates were subjected to Western blot analysis with anti-GFP antibody that recognizes the N-terminal end of GFP-caspase-8A. D) Whole cell lysates were subjected to Western blot analysis with anti-caspase-8 antibody that recognizes the C-terminal end of GFP-caspase-8A. E) Both the inactive C377S and Y465E GFP-caspase-8A mutants failed to undergo cleavage at D391, the essential step of the activation of caspase-8. F-H) HEK293 cells were transfected with GFP-caspase-8B (F), Myc-caspase-8B (G), GFP-caspase-8A (H) WT or mutants for 24 hours. Whole cell lysates were subjected to Western blot analysis with anti-GFP antibody that recognizes the N-terminal end of GFP-caspase-8 and anti-Myc antibody that recognizes the C-terminal end of GFP-caspase-8 (active caspase-8).</p

Predicted SFK tyrosine phosphorylation sites in caspase-8.

<p>Predicted SFK tyrosine phosphorylation sites in caspase-8.</p

Y465 phosphomimetic modification of caspase-8A is necessary but not sufficient for Src activation.

<p>A) HEK293 cells were co-transfected with Y465E GFP-caspase-8A mutant and Y527F Src for 24 hours. Whole cell lysates were subjected to Western blot analysis with an anti-phosphotyrosine antibody (non-reactive to glutamic acid residue in phosphomimetic mutant) and an anti-GFP antibody. Y465E of GFP-caspase-8A was tyrosine phosphorylated. B) HEK293 cells were co-transfected with Y465E GFP-caspase-8A mutant and Y527F Src for 24 hours. GFP-caspase-8A IP was sent for LC-MS/MS analysis. C) This is a schematic figure showing the location of tyrosine residues that we mutated. D, E) We transfected HEK293 cells with Y527F Src and various GFP-caspase-8A mutants for 24 hours. Whole cell lysates were subjected to Western blot analysis with pY416Src antibody and Src antibody. N = 4, <i>p</i> = 0.001 (Y465E vs Y397E/Y465F); <i>p</i> = 0.139 (Y465E vs Y397F/Y465E). F, G) We transfected HEK293 cells with Y527F Src and pEGFP empty plasmid or pEGFP fusion plasmid with WT or various mutants of GFP-caspase-8 for 24 hours. We then immunoprecipitated transfected avian Src and resolve the immunoprecipitate on SDS-PAGE for Western blot analysis. N = 3, <i>p</i> = 0.04.</p