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  • Revisiting the multisite phosphorylation that produces the M-phase supershift of key mitotic regulators.

Revisiting the multisite phosphorylation that produces the M-phase supershift of key mitotic regulators.

Molecular biology of the cell (2022-08-18)
Tan Tan, Chuanfen Wu, Boye Liu, Bih-Fang Pan, David H Hawke, Zehao Su, Shuaishuai Liu, Wei Zhang, Ruoning Wang, Sue-Hwa Lin, Jian Kuang
ABSTRACT

The term M-phase supershift denotes the phosphorylation-dependent substantial increase in the apparent molecular weight of numerous proteins of varied biological functions during M-phase induction. Although the M-phase supershift of multiple key mitotic regulators has been attributed to the multisite phosphorylation catalyzed by the Cdk1/cyclin B/Cks complex, this view is challenged by multiple lines of paradoxical observations. To solve this problem, we reconstituted the M-phase supershift of Xenopus Cdc25C, Myt1, Wee1A, APC3, and Greatwall in Xenopus egg extracts and characterized the supershift-producing phosphorylations. Our results demonstrate that their M-phase supershifts are each due to simultaneous phosphorylation of a considerable portion of S/T/Y residues in a long intrinsically disordered region that is enriched in both S/T residues and S/TP motifs. Although the major mitotic kinases in Xenopus egg extracts, Cdk1, MAPK, Plx1, and RSK2, are able to phosphorylate the five mitotic regulators, they are neither sufficient nor required to produce the M-phase supershift. Accordingly, inhibition of the four major mitotic kinase activities in Xenopus oocytes did not inhibit the M-phase supershift in okadaic acid-induced oocyte maturation. These findings indicate that the M-phase supershift is produced by a previously unrecognized category of mitotic phosphorylation that likely plays important roles in M-phase induction.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Okadaic Acid, Prorocentrum sp., Okadaic Acid, CAS 78111-17-8, is a highly potent inhibitor of protein phosphatase 1 (IC₅₀ = 10-15 nM) and 2A (IC₅₀ = 0.1 nM).
Sigma-Aldrich
Anti-phospho-Histone H1 Antibody, Upstate®, from rabbit
Sigma-Aldrich
Staurosporine from Streptomyces sp., ≥98% (HPLC), film
Sigma-Aldrich
IgG from rabbit serum, technical grade, ≥80% (SDS-PAGE), buffered aqueous solution
Sigma-Aldrich
IgG from mouse serum, technical grade, ≥80% (SDS-PAGE), buffered aqueous solution