Tyrosine phosphorylation of p97 regulates transitional endoplasmic reticulum assembly in vitro

Tyrosine phosphorylation of p97 regulates transitional endoplasmic reticulum assembly in vitro

^*Department of Anatomy and Cell Biology, McGill University, Montreal, QC, H3A 2B2, Canada; ^‡Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724; ^§Department of Medicine, McGill University, Montreal, QC, Canada; and ^¶Département de Pathologie et Biologie Cellulaire, Faculté de Médecine, Université de Montréal, Montréal, QC, H3C 3J7, Canada

Edited by Marilyn Gist Farquhar, University of California, San Diego, CA, and approved September 25, 2000 (received for review June 16, 2000)

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.

Footnotes

↵ † These authors contributed equally to this work.
↵ ‖ To whom reprint requests should be addressed. E-mail: Bergeron{at}med.mcgill.ca.
This paper was submitted directly (Track II) to the PNAS office.
Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073/pnas.240278097.
Article and publication date are at www.pnas.org/cgi/doi/10.1073/pnas.240278097
Abbreviations:

ER,

endoplasmic reticulum;

tER,

transitional endoplasmic reticulum;

LDM,

low-density microsomes;

PTPase,

protein-tyrosine phosphatase