Finely tuned regulation of cytoplasmic retention of Xenopus nuclear factor 7 by phosphorylation of individual threonine residues.
Xenopus nuclear factor 7 (xnf7) is a maternal gene product that functi ons in dorsal/ventral patterning of the embryo. The xnf7 protein is stored in the oocyte nucleus germinal vesicle in a hypophosphorylated state. At oocyte maturation, xnf7 is hyperphosphorylated and released into the cytoplasm, where it is anchored until the midblastula stage, where it is dephosphorylated and enters the nucleus. We demonstrated that cytoplasmic anchoring of xnf7 was regulated by changes in the phosphorylation status of four threonines within two sites, site 1 (Thr-103) and site 2 (Thr-209, Thr-212, and Thr-218), which function in an additive manner. A mutant form of xnf7 (xnf7thr-glu) in which the threonines at sites 1 and 2 were mutated to glutamic acids to mimic a permanent state of phosphorylation was retained in the cytoplasm in oocytes and embryos through the gastrula stage. The cytoplasmic form of xnf7 was detected in a large 670-kDa protein complex probably consisting of xnf7 and several other unknown protein components. Anchoring of xnf7 was not dependent on association with either microtubule or microfilament components of the cytoskeleton, since treatment with cytochalasin B and nocodazole did not affect cytoplasmic retention. Both wild-type xnf7 and xnf7thr-glu form dimers in the yeast two-hybrid system; however, homodimerization was not required for cytoplasmic retention. We suggest that the cytoplasmic retention of xnf7 depends on the phosphorylation state of the protein whereas the cytoplasmic anchoring machinery appears to be constitutively present in oocytes and throughout development until the gastrula stage.
Shou
W
W
Department of Molecular Genetics, University of Texas, Houston 77030, USA.
Li
X
X
Wu
C
C
Cao
T
T
Kuang
J
J
Che
S
S
Etkin
L D
LD
D000595:Amino Acid Sequence; D000818:Animals; D003593:Cytoplasm / Q000378:metabolism; D004268:DNA-Binding Proteins; D004527:Egg Proteins; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D009865:Oocytes / Q000166:cytology / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D013912:Threonine / Q000378:metabolism; D014981:Xenopus / Q000378:metabolism*; D029867:Xenopus Proteins
D004268:DNA-Binding Proteins; D004527:Egg Proteins; D009687:Nuclear Proteins; D010750:Phosphoproteins; D029867:Xenopus Proteins; C071551:XNF7 protein, Xenopus; D013912:Threonine