Fig. 4.

Multiple activities contribute to Emi2 stability and function in CSF extract. (A) Emi2 is destroyed in Δ90 mitotic extract in a CaMKII-independent manner, and Ca²⁺ addition stimulates its rate of destruction. Radiolabeled IVT Emi2 variants were incubated in Δ90 extract with or without Ca²⁺ addition and with or without 281–309 peptide. Emi2 stability and gel mobility were monitored by autoradiography, and CaMKII activation was detected by immunoblotting. The phospho-CaMKII blot is representative of all ±Ca²⁺ pairs except those involving the 281–309 peptide, for which no signal for phospho-CaMKII was detected. (B) Phosphatase inhibition in CSF extract causes cyclin B destruction independent of CaMKII activation and Emi2 destruction. CSF extracts were stimulated with either Ca²⁺ or okadaic acid with or without 281–309 peptide. The stability and gel mobility of radiolabeled IVT Emi2 were monitored by autoradiography. Destruction of cyclin B and activation of CaMKII were monitored by immunoblotting. (C) Emi2 destruction can be induced in okadaic acid-treated CSF extracts, which requires CaMKII and the ₁₉₂RSST and ₃₂DSGYSDS motifs of Emi2. CSF extracts previously stimulated to destroy cyclin by phosphatase inhibition with or without 281–309 peptide were further stimulated by Ca²⁺ addition. Stability and gel mobility of radiolabeled IVT Emi2 variants were monitored by autoradiography.