title: Tyrosine phosphorylation of p97 regulates transitional endoplasmic reticulum assembly in vitro.
abstract: The ATPase associated with different cellular activities family member p97, associated p47, and the t-SNARE syntaxin 5 are necessary for the cell-free reconstitution of transitional endoplasmic reticulum (tER) from starting low-density microsomes. Here, we report that membrane-associated tyrosine kinase and protein-tyrosine phosphatase (PTPase) activities regulate tER assembly by stabilizing (PTPase) or destabilizing (tyrosine kinase) p97 association with membranes. Incubation with the PTPase inhibitor bpV(phen) inhibited tER assembly coincident with the enhanced tyrosine phosphorylation of endogenous p97 and its release from membranes. By contrast, the tyrosine kinase inhibitor, genistein, promoted tER formation and prevented p97 dissociation from membranes while increasing p97 association with the t-SNARE syntaxin 5. Purification of the endogenous tyrosine kinase activity from low-density microsomes led to the identification of JAK-2, whereas PTPH1 was identified as the relevant PTPase. The p97 tyrosine phosphorylation state is proposed to coordinate the assembly of the tER as a regulatory step of the early secretory pathway.
authors.affiliation: Department of Anatomy and Cell Biology, McGill University, Montreal, QC, H3A 2B2, Canada.
authors.forename: C
authors.initials: C
authors.lastname: Lavoie
authors.forename: E
authors.initials: E
authors.lastname: Chevet
authors.forename: L
authors.initials: L
authors.lastname: Roy
authors.forename: N K
authors.initials: NK
authors.lastname: Tonks
authors.forename: A
authors.initials: A
authors.lastname: Fazel
authors.forename: B I
authors.initials: BI
authors.lastname: Posner
authors.forename: J
authors.initials: J
authors.lastname: Paiement
authors.forename: J J
authors.initials: JJ
authors.lastname: Bergeron
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D002474:Cell-Free System; D004721:Endoplasmic Reticulum / Q000648:ultrastructure*; D009687:Nuclear Proteins / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D051381:Rats; D014443:Tyrosine / Q000378:metabolism*
chemical_list: D009687:Nuclear Proteins; D014443:Tyrosine; D000251:Adenosine Triphosphatases; C404942:p97 ATPase