In contrast, bipolar spindles remained stable in control extracts for the duration of the experiment (90 min). MAP kinase signaling was required. To do this, egg extracts made up of duplicated sperm chromosomes were cycled into metaphase in the presence or absence of the pharmacological MEK inhibitor U0126. U0126 inhibits MEK1/2 and not other MAP kinase family members or related kinases (Davies et al., 2000). As expected, the addition of 50 M U0126 strongly inhibited MAP kinase activation but experienced no detectable effect on either Cdc2 or a related MAP kinase family member, JNK1 (Fig. 1 A). Next, spindle assembly was monitored in control- and U0126-treated extracts by epifluorescence. By 60C75 min, metaphase spindles were formed in control extracts (Fig. 1 B, i, and 1 C). In contrast, the assembly of metaphase spindles was completely blocked in the absence of MAP kinase activation (Fig. 1 B, ii, iii, and iv). Typically, we observed three defective spindle phenotypes in U0126-treated extracts: monastral structures without condensed chromatin, open fan-like microtubule structures that are loosely associated with unorganized condensed chromatin, and half-spindleClike structures containing compact chromatin body. The relative percentage for each of these phenotypes is usually shown in Fig. 1 C. Open in a separate window Physique 1. The MEK inhibitor UO126 blocks spindle assembly in CSF-arrested egg extracts Upon activation Sodium succinate of the MAP kinase cascade, p90 Rsk is usually phosphorylated and activated directly by MAP kinase during oocyte maturation (Gross et al., 2000; Kalab et al., 1996) and at mitosis in egg extracts (Bhatt and Ferrell, 1999). Therefore, we tested whether MAP kinase regulation of spindle assembly was exerted through p90 Rsk. Since two closely related Rsk isoforms, Rsk1 and Rsk2, are present and active in eggs (Bhatt and Ferrell, 2000), we used specific Rsk1 and Rsk2 antibodies to sequentially immunodeplete both proteins from CSF-arrested egg extracts. As indicated by immunoblot analysis for Rsk1 and Rsk2, both proteins were quantitatively removed ( 95%) compared with mock-depleted extracts (Fig. 2 D) without affecting endogenous levels of active MAP kinase (Fig. 2 D) or Cdc2 activity (data not shown). Interestingly, in the absence of p90 Rsk, spindle assembly was not compromised (Fig. 2 E): both spindle appearance and the efficiency of spindle formation were comparable in mock- and Rsk1/2-depleted extracts (observe Fig. 2 story). Thus, we conclude that p42 MAP kinase, not p90 Rsk, is required for directly regulating spindle assembly in egg extracts. Depletion of p42 MAP kinase prospects to an increase in the length and polymerization of microtubules in M phase egg extracts Contrary to the static appearance of bipolar spindles, spindle microtubules Sodium succinate are very dynamic with a turnover rate of 60C90 s (Saxton et al., 1984). Therefore, we asked whether MAP kinase might play a role in regulating microtubule dynamics. To address this, we immunodepleted endogenous p42 MAP kinase (96%) from CSF-arrested egg extracts (Fig. 3 A) and measured the length and polymerization of microtubules using an aster Rabbit polyclonal to HER2.This gene encodes a member of the epidermal growth factor (EGF) receptor family of receptor tyrosine kinases.This protein has no ligand binding domain of its own and therefore cannot bind growth factors.However, it does bind tightly to other ligand-boun assay. As obvious in Fig. 3 B, microtubule asters were markedly larger in CSF-arrested extracts depleted of MAP kinase compared with mock-depleted extracts. Precisely, a 24% increase in mean aster radius was Sodium succinate measured over three impartial experiments (Table I). Furthermore, the average total fluorescence intensity/aster increased 30% in MAP kinaseCdepleted extracts compared with mock-depleted extracts, indicative of an increase in microtubule polymerization (Table I). Consistent with this, an increase in tubulin was observed in pelleted microtubules from CSF-arrested extracts depleted of MAP kinase activity (Fig. 3 C). Importantly, the addition of recombinant (his)6-tagged MAP kinase protein to depleted extracts restored MAP kinase activity (Fig. 3 A) and rescued the effects on microtubule polymerization and microtubule length (Fig. 3, B and C, and Table I). Together, our Sodium succinate data support a role for MAP kinase in.