Nuclear localization of an O-glycosylated protein phosphotyrosine phosphatase from human cells.
Histochemical staining of immunoprecipitates of p65, a component of human M phase-promoting factor, identified the molecule as having phosphatase activity. The enzyme, purified 3400-fold from mitotic cell extracts by (NH4)2SO4 precipitation, DEAE chromatography, and immunoaffinity chromatography on immobilized anti-p65 IgG, was inhibited by Zn2- and Na3VO4 but not NaF or beta-glycerophosphate; 32P-labeled poly(Glu, Tyr) was more efficiently dephosphorylated than phosphorylated histone or phosphorylase a. Indirect immunofluorescence showed most of the phosphatase to be localized in the nucleus of interphase cells, with a fine, granular distribution unaltered by detergent extraction; in mitotic cells, p65 was localized on chromosomes. ELISA of subcellular fractions confirmed this localization. Immunoreactive p65 was recovered from immobilized wheat germ agglutinin (WGA) upon elution with N-acetylglucosamine; similarly, WGA recognized immunoaffinity-purified p65 on blots. Alkaline hydrolysis of blotted protein prevented WGA binding, indicating that phosphatase p65, like a small group of other nuclear proteins, contains O-linked carbohydrate terminating in N-acetylglucosamine.
Meikrantz
W
W
Department of Molecular and Cell Biology, Pennsylvania State University, University Park 16802.
Smith
D M
DM
Sladicka
M M
MM
Schlegel
R A
RA
D000117:Acetylglucosamine / Q000032:analysis; D002460:Cell Line; D002467:Cell Nucleus / Q000201:enzymology*; D002875:Chromosomes / Q000201:enzymology; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006023:Glycoproteins / Q000302:isolation & purification; D006801:Humans; D008938:Mitosis; D010749:Phosphoprotein Phosphatases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D010750:Phosphoproteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification*; D011233:Precipitin Tests; D017027:Protein Tyrosine Phosphatases; D013347:Subcellular Fractions / Q000201:enzymology; D013379:Substrate Specificity
D006023:Glycoproteins; D010750:Phosphoproteins; D010749:Phosphoprotein Phosphatases; D017027:Protein Tyrosine Phosphatases; D000117:Acetylglucosamine