Caspase-3-Dependent Cleavage of the Glutamate-L-Cysteine Ligase Catalytic Subunit during Apoptotic Cell Death.
Apoptotic cell death is usually accompanied by activation of a family of cysteine proteases termed caspases. Caspases mediate the selective proteolysis of multiple cellular targets often resulting in the disruption of survival pathways. Intracellular levels of the antioxidant glutathione (GSH) are an important determinant of cellular susceptibility to apoptosis. The rate-limiting step in GSH biosynthesis is mediated by glutamate-L-cysteine ligase (GCL), a heterodimeric enzyme consisting of a catalytic (GCLC) and a modifier (GCLM) subunit. In this report we demonstrate that GCLC is a direct target for caspase-mediated cleavage in multiple models of apoptotic cell death. Mutational analysis revealed that caspase-mediated cleavage of GCLC occurs at Asp(499) within the sequence AVVD(499)G. GCLC cleavage occurs upstream of Cys(553), which is thought to be important for association with GCLM. GCLC cleavage is accompanied by a rapid loss of intracellular GSH due to caspase-mediated extrusion of GSH from the cell. However, while GCLC cleavage is dependent on caspase-3, GSH extrusion occurs by a caspase-3-independent mechanism. Our identification of GCLC as a target for caspase-3-dependent cleavage during apoptotic cell death suggests that this post-translational modification may represent a novel mechanism for regulating GSH biosynthesis during apoptosis.
Franklin
Christopher C
CC
Department of Pathology, University of Washington, Seattle, Washington 98195-7470, USA. cfrankli@u.washington.edu
Krejsa
Cecile M
CM
Pierce
Robert H
RH
White
Collin C
CC
Fausto
Nelson
N
Kavanagh
Terrance J
TJ
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D020134:Catalytic Domain; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D005721:Glutamate-Cysteine Ligase / Q000235:genetics / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D019169:Jurkat Cells; D051379:Mice; D011956:Receptors, Cell Surface / Q000502:physiology; D013997:Time Factors; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D011956:Receptors, Cell Surface; D014409:Tumor Necrosis Factor-alpha; C505407:CASP3 protein, human; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione