Volume 120, Issue 1, Pages 79-88 (January 2001) Hierarchical cleavage of focal adhesion kinase by caspases alters signal transduction during apoptosis of intestinal epithelial cells  Johannes Grossmann, Monika Artinger, Adam W. Grasso, Hsing-Jien Kung, Jürgen Schölmerich, Claudio Fiocchi, Alan D. Levine  Gastroenterology  Volume 120, Issue 1, Pages 79-88 (January 2001) DOI: 10.1053/gast.2001.20879 Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 1 Cleavage of FAK during IEC apoptosis. Cytosol was extracted from epithelial cells at indicated times after induction of DICD and analyzed for FAK by Western blot. Intact FAK (p125) is strongly expressed in human IECs and undergoes 2 sequential cleavages as apoptosis proceeds. The initial cleavage (cleavage I) after 30–45 minutes yields fragments of 94/92 kilodaltons, followed by a second cleavage (cleavage II) after 120–180 minutes to form an 84-kilodalton fragment. Data shown are representative of at least 3 identical experiments. Gastroenterology 2001 120, 79-88DOI: (10.1053/gast.2001.20879) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 2 C-terminal fragment of FAK generated during cleavage I. Cytosol was extracted as described in Figure 1 and probed with an antibody against the C-terminal domain of FAK. As the p94/92 N-terminal fragments are generated during cleavage I of FAK (compare with Figure 1), a corresponding 34-kilodalton C-terminal FAK fragment can be detected. Background signals are enhanced because of the long exposure of the film necessary to detect this fragment (p34). Gastroenterology 2001 120, 79-88DOI: (10.1053/gast.2001.20879) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 3 Fate of PYK-2 during IEC apoptosis. PYK-2 (112 kilodaltons) is expressed in human IECs. During DICD of human IECs, this component of the integrin-associated signaling complex remains uncleaved. Gastroenterology 2001 120, 79-88DOI: (10.1053/gast.2001.20879) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 4 Sequential cleavage of FAK is preserved in vitro. Preapoptotic IEC cytosol was extracted 5 minutes after isolation, followed by in vitro incubation of the cytosol at 37°C. The cytosol was assessed for cleavage of FAK at different times. Note that the sequential cleavage of FAK is preserved in the cytosol, although it proceeds at a faster rate. Gastroenterology 2001 120, 79-88DOI: (10.1053/gast.2001.20879) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 5 Inhibition of (A) FAK cleavage I and (B) FAK cleavage II by caspase inhibitors. Preapoptotic IEC cytosol was incubated with the caspase-3 family inhibitor DEVD-CHO or the caspase-1 family inhibitor YVAD-CHO at different concentrations immediately after isolation (cleavage I) or after the first cleavage of FAK had occurred (i.e., after 30 minutes of in vitro incubation, cleavage II). The effect of these inhibitors on FAK cleavage was assessed by Western blot analysis 120 minutes after the incubation was initiated and quantitated by densitometry. The 100-fold higher sensitivity of both cleavages to DEVD-CHO shows that both cleavages of FAK are mediated by members of the caspase-3 family (P < 0.01). Gastroenterology 2001 120, 79-88DOI: (10.1053/gast.2001.20879) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 6 FAK cleavage is mediated by 2 distinct proteases. Different protease inhibitors were added in vitro to preapoptotic IEC cytosol and incubated for 120 minutes at 37°C. The effect on FAK cleavage was assessed by Western blot analysis. TLCK and EDTA selectively block the second cleavage of FAK. Therefore, FAK cleavage is mediated by 2 distinct proteases. Complete inhibition of FAK cleavage by the cysteine protease inhibitor iodacetamide and the nonselective caspase inhibitor ZVAD-FMK confirms that FAK cleavage during apoptosis is mediated by caspases. Gastroenterology 2001 120, 79-88DOI: (10.1053/gast.2001.20879) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 7 Proteases cleaving FAK show characteristics of caspase-3 and -6. Preapoptotic IEC cytosol was incubated immediately or after cleavage I, as described in Figure 5, with graded concentrations of (A) Zn2+ or (B) VEID-FMK. Cleavage I is less sensitive to inhibition by Zn2+, a characteristic feature of caspase-3, and cleavage II is inhibited by nanomolar concentrations of VEID-FMK, a highly specific inhibitor of caspase-6 (P < 0.02). Gastroenterology 2001 120, 79-88DOI: (10.1053/gast.2001.20879) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 8 Activation of caspase-7 before activation of caspase-3. IEC cytosol was extracted at the indicated times after detachment, and Western blot analyses for (A) caspase-7 (34-kilodalton proenzyme) and (B) caspase-3 (32-kilodalton proenzyme) were performed. Both caspases are expressed as inactive proenzymes in normal human IECs and become activated as IECs undergo detachment-induced apoptosis. Whereas the p20 subunit of activated caspase-7 is first observed after 15 minutes, the p17 subunit of activated caspase-3 is detectable 30–45 minutes after detachment, demonstrating that caspase-3 activation is coincident with FAK cleavage I (see Figure 1). Gastroenterology 2001 120, 79-88DOI: (10.1053/gast.2001.20879) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 9 (A) The p94/p92 and p84 cleavage products of FAK are dephosphorylated during apoptosis. IEC cytosol was extracted at times indicated after detachment. After immunoprecipitation for FAK, proteins were separated by 6% SDS-PAGE and electrotransferred. The membrane was probed with HRP-conjugated antiphosphotyrosine. Although intact FAK and the p94/p92 fragments initially remain phosphorylated, later during apoptosis the p94/p92 and p84 fragments are not detected by the antiphosphotyrosine antibody. Equal amounts of immunoprecipitated FAK and FAK fragments were confirmed by reprobing the membranes with anti-FAK antibody (data not shown). (B) Altered FAK-associated protein phosphorylation patterns are observed in vitro during apoptosis. IEC cytosol was immunoprecipitated for FAK as described above, and a kinase assay was performed using [γ-32P]ATP as a substrate. Intact FAK as well as the FAK fragments incorporates [γ-32P]ATP, suggesting that the FAK kinase domain remains intact during FAK cleavage. Noticeable changes in the pattern of [γ-32P]ATP incorporation are evident in the proteins that co-immunoprecipitated with FAK. Both “gain of function” (solid arrowheads) and “loss of function” (open arrowheads) modifications are indicated. Gastroenterology 2001 120, 79-88DOI: (10.1053/gast.2001.20879) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 10 The p94/p92 and p84 cleavage products of FAK remain associated with the p85 subunit of PI 3-K. IEC cytosol was extracted at times indicated after detachment. (A) After immunoprecipitation for PI 3-K, proteins were separated by 6% SDS-PAGE and electrotransferred. The membrane was probed with mouse anti-FAK. Both intact FAK and the p94/p92 and p84 fragments remain associated with the regulatory subunit of PI 3-K, despite the transient loss of phosphorylation of this protein by FAK. Equal amounts of immunoprecipitated PI 3-K were confirmed by reprobing the membranes with anti-PI 3-K antibody (data not shown). (B) Similar results were obtained when the same cytosols were conversely immunoprecipitated with an antibody that recognizes intact and cleaved FAK, and the membrane was probed with rabbit anti–PI 3-K. Gastroenterology 2001 120, 79-88DOI: (10.1053/gast.2001.20879) Copyright © 2001 American Gastroenterological Association Terms and Conditions