Src kinase phosphorylates Caspase-8 on Tyr380: a novel mechanism of apoptosis suppression

Abstract

We identified Caspase-8 as a new substrate for Src kinase. Phosphorylation occurs on Tyr380, situated in the linker region between the large and the small subunits of human Procaspase-8, and results in downregulation of Caspase-8 proapoptotic function. Src activation triggers Caspase-8 phosphorylation on Tyr380 and impairs Fas-induced apoptosis. Accordingly, Src failed to protect Caspase-8-defective human cells in which a Caspase-8-Y380F mutant is expressed from Fas-induced cell death. Remarkably, Src activation upon EGF-receptor stimulation triggers endogenous Caspase-8 phosphorylation and prevents Fas-induced apoptosis. Tyr380 is phosphorylated also in human colon cancers where Src is aberrantly activated. These data provide the first evidence for a direct role of tyrosine phosphorylation in the control of caspases and reveal a new mechanism through which tyrosine kinases inhibit apoptosis and participate in tumor progression.

Figure 1

Src kinase phosphorylates Caspase-8. HEK293 cells were transiently transfected with Caspase-8, and either Src or Abl kinases. All transfections included pEGFP-N1-CrmA to prevent the induction of apoptosis due to Caspase-8 overexpression. Total protein extracts or immunoprecipitated Caspase-8 were separated by SDS–PAGE and immunoblotted with specific antibodies. The ^* points to a portion of Caspase-8 that corresponds to the protein translated from the Met43 (Medema et al, 1997). On total immunoblots Src and Caspase-8 migrate at the same Mw, as shown by the arrow. c-Jun has been used as a control substrate of Abl.

Figure 2

Src kinase activity modulates Fas-induced apoptosis and Caspase-8 activity. (A) HeLa cells were transiently transfected with SrcY527F or with the empty vector as control. Immunoprecipitated Caspase-8 was separated by SDS–PAGE gel and analyzed by immunoblotting with anti-phosphotyrosine and with anti-Caspase-8 antibodies. The arrow indicates Caspase-8. (B) At 24 h after transfection, HeLa cells, transiently transfected with the indicated constructs, were treated with 250 ng/ml anti-Fas and 1 μg/ml cycloheximide. Apoptosis was determined by counting Hoechst-stained fragmented nuclei in GFP-positive cells. The data are presented as the mean value±s.d. of five independent experiments. (C) HeLa cells were transfected with the indicated expression constructs. Apoptosis was induced as in Figure 2B and Caspase-8 activity was analyzed in protein extract by measuring the hydrolysis of the Caspase-8 substrate Ac-IETD-pNA. Data are presented as the mean value±s.d. of three independent experiments.

Figure 3

Src kinase phosphorylates Caspase-8 mainly on Tyr380. (A) Myc-Caspase-8 has been immunoprecipitated from extracts prepared from HEK293 cells transiently cotransfected with Myc-Caspase-8, SrcY527F and CrmA. Immunoprecipitated Caspase-8 has been recovered from SDS–PAGE upon Coomassie staining and the protein processed for MS. The picture shows the product ion spectrum of the oxidated form of the phosphopeptide GIPVETDSEEQPpYLEM(ox)DLSSPQTR (precursor ion MH3+=939.6 m/z). The region of the spectrum where the ions corresponding to the sequence QP pY LEM(ox) is enlarged. Peaks corresponding to y′ series are labeled. (B) HEK293 cells were cotransfected with the indicated constructs always along with CrmA. Total protein extracts or immunoprecipitated Caspase-8 were separated by SDS–PAGE and immunoblotted with specific antibodies. (C) Alignment of the putative linker region between the p18 and the p10 subunit of Caspase-8 in different species. Tyrosine residues corresponding toTyr380 on the human sequence have been highlighted in bold.

Figure 4

Src kinase phosphorylates Caspase-8 on Tyr380 and modulates its processing and activity in yeast. (A) Protein extracts from Jurkat cells stimulated to undergo apoptosis with anti-Fas antibodies, and protein extracts from S. pombe at different times of Caspase-8 expression induction, have been separated by SDS–PAGE and Caspase-8 revealed by immunoblotting with specific antibodies. The arrows point to the entire protein, p55, as well as to the processing products p43 and p18. (B) Extracts from S. pombe transfected with Caspase-8-wt in the presence or not of Src, at different times of induction, have been processed for SDS–PAGE and immunoblotting with specific antibodies. (C) Caspase-8 activity from S. pombe extracts at 13 h of induction was measured by the hydrolysis of the Caspase-8 substrate Ac-IETD-AMC. The differences between Caspase-8-wt±Src (^*) are statistically significant by t-test (^*P=0.02). (D) Growth curves of S. pombe cells expressing Src from the inducible nmt1 promoter of the pRSP vector and Caspase-8 wt or Y380F from the inducible nmt1 promoter of the pNU vector. The y-axis is logarithmic. (E) The histogram represents the number of cells, at 36 h after induction of expression of proteins. Each bar represents the mean value±s.d. (n=8). The differences in the cell number between Caspase-8-wt±Src (^*) and between Caspase-8-wt+Src and Caspase-8-Y380F+Src (#) are statistically significant by t-test (^*P=0.02, ^#P=0.0003).

Figure 5

Phosphorylation on Tyr380 modulates the activity of Caspase-8 and protects human cells from Fas-induced apoptosis. (A) HeLa cells were transiently transfected with SrcY527F, or with empty vector, and Procaspase-8 dimerization was triggered by incubation of protein extracts in the presence of Sodium Citrate. Active dimerized Procaspase-8 was revealed by Biotin-VAD-fmk labeling, immunoprecipitation with agarose-conjugated streptavidin beads and immunoblotting. (B) HeLa cells transfected as indicated were stimulated to undergo apoptosis with 250 ng/ml α-Fas plus 1 μg/ml cycloheximide for 2.5 and 5 h. Total lysates were separated by SDS–PAGE and immunoblotted with specific anti-Caspase-8 antibodies. (C) Active Caspase-8 from 10⁷cells, transfected as in (A) and stimulated with 500 ng/ml α-Fas plus 1 μg/ml cycloheximide for 4 h, was labeled with Biotin-VAD-fmk, immunoprecipitated with agarose-conjugated streptavidin beads and immunoblotted as indicated. (D) Caspase-8-deficient Jurkat I9.2 cell line was transiently transfected with Caspase-8-wt or Caspase-8-Y380F in the presence or not of SrcY527F, together with pEGFP-N1-spectrin. At 12 h after transfection, cells were incubated with 500 ng/ml anti-Fas antibodies for 8 h. Apoptosis was measured by flow-cytometry upon propidium iodide staining of the GFP-positive population. The data are presented as the mean value±s.d. of five independent experiments. The differences between Caspase-8-wt+Src and Caspase-8 (^*), and between Caspase-8-wt+Src and Caspase-8-Y380F+Src (^#) are statistically significant by t-test (^*P=0.04, ^#P=0.01).

Figure 6

EGF triggers Caspase-8 tyrosine phosphorylation and protects cells from Fas-induced apoptosis. (A) HeLa cells were serum-deprived for 24 h and then treated with 100 ng/ml EGF for 15 min. Immunoprecipitated Caspase-8 was separated by SDS–PAGE gel and analyzed by immunoblotting with anti-phosphotyrosine and with anti-Caspase-8 antibodies. Src activation was revealed by immunoblotting with an antibody that selectively recognizes active Src, upon immunoprecipitation with anti-phosphotyrosine antibodies (B) HeLa cells stably transfected with Src-Y527F-Kin⁻ or with empty vector as control have been serum starved for 24 h and stimulated to undergo apoptosis with 500 ng/ml anti-Fas antibody, 1 μg/ml CHX, in the presence or not of 100 ng/ml EGF. Apoptotic cells have been identified by Hoechst-stained fragmented nuclei. The rate of apoptosis has been calculated at different times of stimulation. The data are presented as the mean value±s.d. of six independent experiments.

Figure 7

Caspase-8 is phosphorylated on Tyr380 in colon cancer. (A) Protein extracts were prepared from human colorectal carcinoma and corresponding normal samples. Total proteins were separated by SDS–PAGE and immunoblotted with specific antibodies. (B) The pool of tyrosine phosphorylated proteins was immunoprecipitated with anti-phosphotyrosine antibodies and Caspase-8 and active Src detected by immunoblotting with specific antibodies. (C) Caspase-8 was immunoprecipitated and tyrosine phosphorylation was revealed by immunoblotting with anti-phosphotyrosine antibodies. Src was immunoprecipitated and its activity was revealed by immunoblotting with specific antibodies. (D) Caspase-8 was immunoprecipitated with anti-Caspase-8 antibodies and immunoblotted with phospho-Casp8-(Y380) antibody. (E) Extracts from DLD-1/TRAIL-R cells transiently transfected with the indicated constructs were immunoblotted as shown. (F) DLD-1/TRAIL-R cell line was transiently transfected with Caspase-8-wt or Caspase-8-Y380F together with pEGFP-N1-spectrin. At 24 h after transfection, cells were incubated with 500 ng/ml anti-Fas plus 1 μg/ml cycloheximide for 3 h. Apoptosis was measured by flow-cytometry upon propidium iodide staining of the GFP-positive population. The data are presented as the mean value±s.d. of three independent experiments (^*P=0.03).