PHOSPHORYLATION OF THE GUANINE-NUCLEOTIDE EXCHANGE FACTOR AND EUKARYOTIC INITIATION-FACTOR-2 BY CASEIN KINASE-II REGULATES GUANINE-NUCLEOTIDE-BINDING AND GDP GTP EXCHANGE/

In mammalian cells, chain initiation factor (eIF) 2 and guanine nucleo tide exchange factor (GEF) play a major role in the regulation of poly peptide chain initiation. Since guanine nucleotide exchange is the rat e-limiting step in the recycling of eIF-2, we examined the effects of phosphorylation of GEF and eIF-2 on guanine nucleotide binding and the rate of GDP/GTP exchange. Phosphorylation of the 82-kDa subunit of GE F in vitro by casein kinase (CK) II results in the stimulation of guan ine nucleotide exchange [Dholakia, J. N., & Wahba, A. J. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 51-54]. CK-II also phosphorylates the beta -subunit of eIF2, but the significance of this phosphorylation has not previously been investigated. In this study we demonstrate that treat ment of CK-II-phosphorylated GEF or eIF-2 with alkaline phosphatase sp ecifically removes more than 85% of the phosphate incorporated into th e factors and alters guanine nucleotide binding to these proteins. In the presence of 1 mM Mg2+, the amount of GTP bound to dephosphorylated GEF is reduced 3.8-fold as compared to that of the CK-II-phosphorylat ed GEF. Rephosphorylation with CK-II restores GTP binding and increase s 4-5-fold the activity of GEF in the exchange of eIF-2-bound GDP for free GTP. On the other hand, the extent of GDP binding to dephosphoryl ated eIF-2 is increased 2.3-fold as compared to that to the isolated e IF-2. The rate of GEF-catalyzed exchange of dephosphorylated eIF-2-bou nd GDP for GTP is approximately 2-fold slower than that with the isola ted eIF-2. These results suggest that phosphorylation of GEF and eIF-2 with CK-II provides a mechanism for the regulation of nucleotide bind ing and GDP/GTP exchange during polypeptide chain initiation.