Expression and functional analysis of Apaf-1 isoforms - Extra WD-40 repeat is required for cytochrome c binding and regulated activation of procaspase-9

University of Michigan, Ann Arbor, Michigan, United States
Journal of Biological Chemistry (Impact Factor: 4.57). 04/2000; 275(12):8461-8. DOI: 10.1074/jbc.275.12.8461
Source: PubMed

ABSTRACT Apaf-1 is an important apoptotic signaling molecule that can activate procaspase-9 in a cytochrome c/dATP-dependent fashion. Alternative splicing can create an NH(2)-terminal 11-amino acid insert between the caspase recruitment domain and ATPase domains or an additional COOH-terminal WD-40 repeat. Recently, several Apaf-1 isoforms have been identified in tumor cell lines, but their expression in tissues and ability to activate procaspase-9 remain poorly characterized. We performed analysis of normal tissue mRNAs to examine the relative expression of the Apaf-1 forms and identified Apaf-1XL, containing both the NH(2)-terminal and COOH-terminal inserts, as the major RNA form expressed in all tissues tested. We also identified another expressed isoform, Apaf-1LN, containing the NH(2)-terminal insert, but lacking the additional WD-40 repeat. Functional analysis of all identified Apaf-1 isoforms demonstrated that only those with the additional WD-40 repeat activated procaspase 9 in vitro in response to cytochrome c and dATP, while the NH(2)-terminal insert was not required for this activity. Consistent with this result, in vitro binding assays demonstrated that the additional WD-40 repeat was also required for binding of cytochrome c, subsequent Apaf-1 self-association, binding to procaspase-9, and formation of active Apaf-1 oligomers. These experiments demonstrate the expression of multiple Apaf-1 isoforms and show that only those containing the additional WD-40 repeat bind and activate procaspase-9 in response to cytochrome c and dATP.

  • Source
    • "The large family of WD40 repeat-containing proteins can be engaged in a broad range of cellular events, such as signal transduction (Parent et al., 2008; Smith et al., 1999), RNA processing complexes (Mitsuzawa et al., 2001), transcriptional regulation (Pickles et al., 2002), cytoskeleton assembly and mitotic spindle formation (Welch et al., 1997), vesicle formation and trafficking (Fath et al., 2007), cell division control (Yu, 2007) and programmed cell death (Benedict et al., 2000). The basic common function of WD40 repeat-containing proteins is to coordinate multi-protein complex assembly, where the repeating units serve as a scaffold for protein interactions. "
    [Show abstract] [Hide abstract]
    ABSTRACT: We identified the WD-repeat-containing protein, WDR36, as an interacting partner of the β isoform of thromboxane A(2) receptor (TPβ) by yeast two-hybrid screening. We demonstrated that WDR36 directly interacts with the C-terminus and the first intracellular loop of TPβ by in vitro GST-pulldown assays. The interaction in a cellular context was observed by co-immunoprecipitation, which was positively affected by TPβ stimulation. TPβ-WDR36 colocalization was detected by confocal microscopy at the plasma membrane in non-stimulated HEK293 cells but the complex translocated to intracellular vesicles following receptor stimulation. Coexpression of WDR36 and its siRNA-mediated knockdown, respectively, increased and inhibited TPβ-induced Gαq signalling. Interestingly, WDR36 co-immunoprecipitated with Gαq, and promoted TPβ-Gαq interaction. WDR36 also associated with phospholipase Cβ (PLCβ) and increased the interaction between Gαq and PLCβ, but prevented sequestration of activated Gαq by GRK2. In addition, the presence of TPβ in PLCβ immunoprecipitates was augmented by expression of WDR36. Finally, disease-associated variants of WDR36 affected its ability to modulate Gαq-mediated signalling by TPβ. We report that WDR36 acts as a new scaffold protein tethering a G-protein-coupled receptor, Gαq and PLCβ in a signalling complex.
    Journal of Cell Science 10/2011; 124(Pt 19):3292-304. DOI:10.1242/jcs.085795 · 5.33 Impact Factor
  • Source
    • "These apparently conflicting results suggest that BCL2L13 may have a different apoptotic role in childhood ALL in comparison to its behavior in the cell lines that were used to characterize its apoptotic functions. One possible mechanism to explain this difference is alternative splicing, which is known to generate both anti-and pro-apoptic variants of apoptosis genes such as Apaf-1 and Livin [12] [13] [14]. The explanation for the finding that high expression of BCL2L13 was associated with inferior treatment response will thus require further study. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Improved treatment of childhood acute lymphoblastic leukemia (ALL) depends on the identification of new molecular markers that are able to predict treatment response and clinical outcome. The development of impaired apoptosis in leukemic cells is one factor that may influence their response to treatment. We investigated the expression of three apoptosis related genes, BCL2L13, CASP8AP2, and Livin, as well as their prognostic significance, in a retrospective study of 90 pediatric ALL patients diagnosed between 1996 and 2007 in Taiwan. Univariant analysis revealed that high expression of BCL2L13 was associated with inferior event-free survival and overall survival (p<0.001 and 0.005, respectively). Multivariate analysis for EFS and OS demonstrated that high expression of BCL2L13 was an independent prognostic factor for childhood ALL in this ethnic group.
    Leukemia research 01/2010; 34(1):18-23. DOI:10.1016/j.leukres.2009.07.023 · 2.69 Impact Factor
  • Source
    • "All reagents for recombinant system were prepared as described (Benedict et al. 2000; Fearnhead et al. 1998; Lin 2006; Rodriguez and Lazebnik 1999; Zou et al. 1999). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Caspase-9 is the protease that mediates the intrinsic pathway of apoptosis, a type of cell death. Activation of caspase-9 is a multi-step process that requires dATP or ATP and involves at least two proteins, cytochrome c and Apaf-1. In this study, we mathematically model caspase-9 activation by using a system of ordinary differential equations (an ODE model) generated by a systems biology tool Simpathica—a simulation and reasoning system, developed to study biological pathways. A rudimentary version of “model checking” based on comparing simulation data with that obtained from a recombinant system of caspase-9 activation, provided several new insights into regulation of this protease. The model predicts that the activation begins with binding of dATP to Apaf-1, which initiates the interaction between Apaf-1 and cytochrome c, thus forming a complex that oligomerizes into an active caspase-9 holoenzyme via a linear binding model with cooperative interaction rather than through network formation. Electronic supplementary material The online version of this article (doi:10.1007/s11693-009-9022-y) contains supplementary material, which is available to authorized users.
    Systems and Synthetic Biology 04/2009; 2(1-2):49-66. DOI:10.1007/s11693-009-9022-y
Show more