Abstract
We have previously identified a 15-kDa interferon-induced protein that is recognized by affinity-purified rabbit polyclonal antibodies against ubiquitin (Haas, A. L., Ahrens, P., Bright, P. M., and Ankel, H. (1987) J. Biol. Chem. 262, 11315-11323). This ubiquitin cross-reactive protein (UCRP) possesses significant homology to a tandem diubiquitin sequence. Since the biological effects of ubiquitin arise from its covalent ligation to intracellular target proteins, we hypothesized that the multiple cellular responses to inteferon are mediated in part by an analogous conjugation pathway for UCRP. Rabbit polyclonal antibodies specific for UCRP were prepared against homogeneous recombinant protein. Affinity-purified anti-UCRP antibodies detected the induction of UCRP in interferon-beta-treated A549 cells and recognized a group of high molecular weight UCRP conjugates on immunoblots of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-resolved cell extracts. Both free and conjugated UCRP are constitutively present at low levels in untreated cells, suggesting a role for UCRP ligation in normal cellular regulation, and significantly accumulate following interferon treatment. The temporal induction of free UCRP following interferon treatment preceded a delayed increase in UCRP conjugates. Treatment of A549 cells with type I interferons (alpha and beta) strongly induced the expression of free and conjugated UCRP, whereas the response to type II interferon (gamma) was significantly less. A survey of selected cultured cell lines showed differential induction of free versus conjugated UCRP pools in response to interferon. Interferon-beta treatment of A549, MG63, and U937 cells induced high levels of both free and conjugated UCRP, whereas only free UCRP levels increased in Daudi, Namalwa, and K562 cells. These results confirm that UCRP represents a functional ubiquitin homolog participating in a parallel pathway of post-translational ligation and provides a novel mechanism for the response of susceptible cells to the effects of interferon exposure.
Highlights
We have previously identified a 15-kDa interferon- Ubiquitin is one of the most highly conserved eukaryotic induced protein thatis recognized by affinity-purified proteins characterized to date, differing in only three amino rabbit polyclonal antibodies against ubiquitin
Sincethe biological effects of ubiquitin arise from its covalent ligation to intracellular target proteins, we hypothesized that themultiple cellular responses to interferon are mediated in partby an analogous conjugation pathof fundamental regulatory processes through a unique posttranslational modification in which the carboxyl terminus of ubiquitin is covalently ligated to primary amines on a variety of intracellular proteins
The temporal in- A recurring question has been whether ubiquitin ligation duction of free ubiquitin cross-reactive protein (UCRP) following interferon treatment preceded a delayedincreasein UCRPconjugates
Summary
0 1992 by The American Society for Biochemistry and Molecular Biology, Inc. Vol 267, No 11, Issue of April 15, pp. 7806-7813, 1992 Printed in U.S.A. Affinity-purified anti-UCRP antibod- dependent proteolysis has been implicated in the degraies detected the induction of UCRP in interferon-b- dation of transiently short lived proteins, including several treated A549 cells and recognized a group of high nuclear regulatory proteins, such as thep tumor suppressor, molecular weight UCRP conjugateosn immunoblots of cyclin, N/c-myc, and C-fos (4-6).In addition, relatively stable sodium dodecyl sulfate-polyacrylamide gel electrophou-biquitin conjugates to core histones (7, 8) and to plasma resis-resolved cell extracts. Both free and conjugated membrane receptors (9-11) have been identified, suggesting. We provide evidence supporting the posttranslational ligation of UCRP to endogenous cellular proteins and demonstrate that such UCRP-protein conjugates are constitutively present in all cells tested and interferoninducible in a subsetof susceptible cells
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