title: Bak apoptotic function is not directly regulated by phosphorylation.
abstract: During apoptosis, Bak and Bax permeabilize the mitochondrial outer membrane by undergoing major conformational change and oligomerization. This activation process in Bak is reported to require dephosphorylation of tyrosine-108 close to an activation trigger site. To investigate how dephosphorylation of Bak contributes to its activation and conformational change, one-dimensional isoelectric focusing (1D-IEF) and mutagenesis was used to monitor Bak phosphorylation. On 1D-IEF, Bak extracted from a range of cell types migrated as a single band near the predicted isoelectric point of 5.6 both before and after phosphatase treatment, indicating that Bak is not significantly phosphorylated at any residue. In contrast, three engineered 'phosphotagged' Bak variants showed a second band at lower pI, indicating phosphorylation. Apoptosis induced by several stimuli failed to alter Bak pI, indicating little change in phosphorylation status. In addition, alanine substitution of tyrosine-108 and other putative phosphorylation sites failed to enhance Bak activation or pro-apoptotic function. In summary, Bak is not significantly phosphorylated at any residue, and Bak activation during apoptosis does not require dephosphorylation.
authors.affiliation: Molecular Genetics of Cancer Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia.
authors.forename: V H
authors.initials: VH
authors.lastname: Tran
authors.forename: R
authors.initials: R
authors.lastname: Bartolo
authors.forename: D
authors.initials: D
authors.lastname: Westphal
authors.forename: A
authors.initials: A
authors.lastname: Alsop
authors.forename: G
authors.initials: G
authors.lastname: Dewson
authors.forename: R M
authors.initials: RM
authors.lastname: Kluck
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D000970:Antineoplastic Agents / Q000633:toxicity; D017209:Apoptosis / Q000187:drug effects; D002460:Cell Line; D006801:Humans; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D019169:Jurkat Cells; D051379:Mice; D008810:Mice, Inbred C57BL; D008928:Mitochondria / Q000378:metabolism; D008969:Molecular Sequence Data; D010766:Phosphorylation / Q000187:drug effects; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism; D014443:Tyrosine / Q000737:chemistry; D050998:bcl-2 Homologous Antagonist-Killer Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D000970:Antineoplastic Agents; D050998:bcl-2 Homologous Antagonist-Killer Protein; D014443:Tyrosine; D017027:Protein Tyrosine Phosphatases