Role of protein phosphorylation in activation of interferon-stimulated gene factors.

S.K Bandyopadhyay,G.C Sen

doi:10.1016/s0021-9258(18)42707-x

Abstract

Possible involvement of protein phosphorylation in interferon (IFN)-mediated activation of IFN-stimulated gene factor 3 (ISGF3) was investigated. For this purpose, in vivo experiments with specific inhibitors of protein kinases and in vitro experiments with protein phosphatases were carried out. In HeLaM cells, 2-aminopurine, an inhibitor of double-stranded RNA-dependent protein kinase, blocked the induction of ISGF3 gamma subunit but not the activation of ISGF3 alpha subunit. A series of experiments using combinations of protein and RNA synthesis inhibitors and 2-aminopurine indicated that the block elicited by 2-aminopurine was at the level of ISGF3 gamma mRNA synthesis. Activation of ISGF3 alpha, although insensitive to 2-aminopurine, was completely blocked by 10 nM staurosporine, an inhibitor of protein kinase C. On the other hand, even 500 nM staurosporine did not block the induction of ISGF3 gamma. Incubation of cytoplasmic or nuclear extracts of IFN-treated HeLaM cells in vitro with alkaline phosphatase completely eliminated their ability to form the ISGF3 complex but not the ISGF1 complex. Treatment with acid phosphatase, on the other hand, changed the electrophoretic mobility of the ISGF3 complex but did not obliterate it. Complementation experiments revealed that ISGF3 alpha was the alkaline phosphatase-sensitive component of the complex. These results suggest that a protein kinase C-mediated phosphorylation step is involved in ISGF3 alpha activation and a 2-aminopurine-sensitive component is involved in ISGF3 gamma mRNA induction.

Highlights

Dependentprotein kinase, blockedtheinductionof ISGF3-y subunit but notthe activation of ISGF3a subunit
Upon IFN-a treatment, active ISGF3 appears first in the cytoplasm and it translocates to the nucleus where it presumably binds to the ISREs of different IFN-inducible genes and thereby activates their transcription
In our early studies with HeLaM cells, we observed that gene induction by IFN-a required ongoing protein synthesis which could be obviated by pretreating the cells with IFN--y [39, 41]

Summary

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ActD ine. Induction of ISGF3y followed by a treatment with staurosporine, on the other hand, did not obliterate i t s activity (Fig. 3A, lane 9 ). IFN-a induced ISGFB and either CHX or staurosporbilnoecked this inductionprocess (Fig. 3B, lanes 3-5). *. CHX.n induction or ISGF3a activationindividually, it was apparent that ISGF3ywas induced by IFN-n in the presenceof 500 nM. Did not block ISGF3a actinot block ISCF3-y induction for an additional h; lone 6, same as lane 4 except that actinomycin by IFN-a,sincetreatmentwithPMA followed by IFN-a. T h e treatment by itself, did not activate ISGF3a (Fig. 4, lane 5). When cells were treated for 24 hwith PMA to compositions of the mixtures in different lanes are indicated on the completely down-regulate protein kinase C from the cell surfigure

Protein Phosphorylation inIFN Signal Transduction

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Nuclear Extracts

DISCUSSION