Phosphorylation positively regulates DNA binding of the carbon catabolite repressor Cre1 of Hypocrea jecorina (Trichoderma reesei).
Cre1 of the ascomycete Hypocrea jecorina is a Cys(2)His(2) zinc finger DNA-binding protein functioning as regulator for carbon catabolite repression. It represents the functional equivalent of yeast Mig1, known to be negatively regulated by the Snf1-kinase at the nuclear import level. We demonstrate that Cre1 is also a phosphoprotein, and identify Ser(241) within an acidic protein region as phosphorylation target. In contrast to Mig1 phosphorylation is required for DNA binding of Cre1. A S241E mutation mimics phosphorylation, whereas a S241A mutant protein shows phosphorylation-independent DNA binding activity, suggesting that phosphorylation is required to release Cre1 from an inactive conformation involving unphosphorylated Ser(241). Retransformation of a H. jecorina cre1-non functional mutant with Cre1-S241A leads to permanent carbon catabolite repression in cellobiohydrolase I expression. Contrary to Mig1, the amino acid sequence surrounding Ser(241) (HSNDEDD) suggests that phosphorylation may occur by a casein kinase II-like protein. This is supported by a mutation of E244V leading to loss of phosphorylation, loss of DNA binding, and gain of carbon catabolite derepression. Our results imply that the regulation of carbon catabolite repression at the level of DNA binding strongly differs between Saccharomyces cerevisiae and H. jecorina.
Cziferszky
Angela
A
Section for Microbial Biochemistry and Gene Technology, Institute of Chemical Engineering, Technical University of Vienna, Getreidemarkt 9-166, A-1060 Vienna, Austria.
Mach
Robert L
RL
Kubicek
Christian P
CP
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D015152:Blotting, Northern; D015139:Blotting, Southern; D047388:Casein Kinases; D017931:DNA Primers; D004271:DNA, Fungal / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D005656:Fungal Proteins; D015966:Gene Expression Regulation, Fungal / Q000502:physiology; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D014242:Trichoderma / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D011994:Recombinant Proteins; D012097:Repressor Proteins; D012694:Serine; D011494:Protein Kinases; D047388:Casein Kinases