TY - JOUR
T1 - Phosphorylation of a 225-kDa centrosomal component in mitotic CHO cells and sea urchin eggs
AU - Kuriyama, Ryoko
AU - Maekawa, Takami
PY - 1992/10
Y1 - 1992/10
N2 - Components of centrosomes are those among cellular proteins that are phosphorylated at the transition from interphase to mitosis. Using an anti-phosphoprotein antibody (CHO3) directed against isolated mitotic CHO spindles, we identified a 225-kDa centrosomal phosphocomponent in mitotic CHO cells and in cleaving sea urchin eggs. The 225-kDa protein is tightly attached to the centrosome, which allowed us to separate it from other spindle-associated factors by high salt extraction. Phosphorylation of the 225-kDa protein occurred during mitosis. This was shown by isotope labeling on gels as well as by visualization of thiophosphorylated centrosomes with an anti-thiophosphoprotein antibody (M. Cyert, T. Scherson, and M.W. Kirschner, 1988, Dev. Biol. 129, 209) after preincubation with ATP-γ-S in vivo and in vitro. Mitotic spindles isolated from CHO cells retained their ability to phosphorylate the centrosomal component, whereas sea urchin spindles did not, possibly due to loss or inactivation of protein kinase(s) during spindle isolation. The enzyme associated with isolated CHO spindles was extractable by high salt treatment and was capable of phosphorylating many spindle components, including the 225-kDa centrosomal protein of CHO cells and sea urchin embryos. Such high salt extracts contain protein kinases, including cell cycle control protein kinase p34cdc2, suggesting that the enzyme responsible for centrosomal phosphorylation could be p34cdc2 or other downstream mitotic kinases activated by the action of p34cdc2.
AB - Components of centrosomes are those among cellular proteins that are phosphorylated at the transition from interphase to mitosis. Using an anti-phosphoprotein antibody (CHO3) directed against isolated mitotic CHO spindles, we identified a 225-kDa centrosomal phosphocomponent in mitotic CHO cells and in cleaving sea urchin eggs. The 225-kDa protein is tightly attached to the centrosome, which allowed us to separate it from other spindle-associated factors by high salt extraction. Phosphorylation of the 225-kDa protein occurred during mitosis. This was shown by isotope labeling on gels as well as by visualization of thiophosphorylated centrosomes with an anti-thiophosphoprotein antibody (M. Cyert, T. Scherson, and M.W. Kirschner, 1988, Dev. Biol. 129, 209) after preincubation with ATP-γ-S in vivo and in vitro. Mitotic spindles isolated from CHO cells retained their ability to phosphorylate the centrosomal component, whereas sea urchin spindles did not, possibly due to loss or inactivation of protein kinase(s) during spindle isolation. The enzyme associated with isolated CHO spindles was extractable by high salt treatment and was capable of phosphorylating many spindle components, including the 225-kDa centrosomal protein of CHO cells and sea urchin embryos. Such high salt extracts contain protein kinases, including cell cycle control protein kinase p34cdc2, suggesting that the enzyme responsible for centrosomal phosphorylation could be p34cdc2 or other downstream mitotic kinases activated by the action of p34cdc2.
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U2 - 10.1016/0014-4827(92)90084-L
DO - 10.1016/0014-4827(92)90084-L
M3 - Article
C2 - 1397087
AN - SCOPUS:0026725793
VL - 202
SP - 345
EP - 354
JO - Experimental Cell Research
JF - Experimental Cell Research
SN - 0014-4827
IS - 2
ER -