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 nucleotide exchange factor (GEF) play a major role in the regulation of polypeptide chain initiation. Since guanine nucleotide exchange is the rate-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 GEF in vitro by casein kinase (CK) II results in the stimulation of guanine 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 treatment of CK-II-phosphorylated GEF or eIF-2 with alkaline phosphatase specifically removes more than 85% of the phosphate incorporated into the 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-phosphorylated GEF. Rephosphorylation with CK-II restores GTP binding and increases 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 dephosphorylated eIF-2 is increased 2.3-fold as compared to that to the isolated eIF-2. The rate of GEF-catalyzed exchange of dephosphorylated eIF-2-bound GDP for GTP is approximately 2-fold slower than that with the isolated eIF-2.(ABSTRACT TRUNCATED AT 250 WORDS)
Singh
L P
LP
Department of Biochemistry, University of Mississippi Medical Center, Jackson 39216.
Arorr
A R
AR
Wahba
A J
AJ
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D047390:Casein Kinase II; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D015852:Eukaryotic Initiation Factor-2 / Q000302:isolation & purification / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000302:isolation & purification / Q000378:metabolism*; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism*; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D013997:Time Factors
D015852:Eukaryotic Initiation Factor-2; D020662:Guanine Nucleotide Exchange Factors; D046911:Macromolecular Substances; D011506:Proteins; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases