Therefore, p38 MAPK is probably not involved in 3F3/2 antigen phosphorylation. Another important result from our study is the localization of active ERK at the chromosome periphery during prophase and prometaphase, as well as at spindle Capromorelin poles during prophase, metaphase, and anaphase. for the 3F3/2 monoclonal antibody, which recognizes a phosphoepitope that disappears with kinetochore attachment to the spindles, and has PIK3C1 been implicated in the mitotic checkpoint for anaphase onset (Gorbsky and Ricketts, 1993. cell free extracts leads to arrest in G2 and suppression of cyclin B/cdc2 activation (Abrieu et al., 1997; Walter et al., 1997). Taken together, these data suggest that ERK functions positively during meiotic cell division, but in fact negatively regulates mitotic Capromorelin progression in early embryos. Consistent with results from early embryos, previous reports in somatic mammalian cells have shown no activation of ERK during mitosis, as measured by SDS-PAGE gel mobility retardation or in-gel phosphorylation assays (Tamemoto et al., 1992; Edelmann et al., 1996). Ras also appears to remain inactive Capromorelin during mitosis (Taylor and Shalloway, 1996). Nevertheless, mitotic enhancement of Raf-1 activity in cells synchronized by mitotic shake-off or arrested with nocodazole has been reported (Laird et al., 1995; Pathan et al., 1996), and inhibition of c-Src by antibody microinjection blocks mitotic entry (Roche et al., 1995). These data indicate the existence of mitotic mechanisms for activating ERK through known upstream pathway components in somatic cells. In this study, we examined the cellular localization of active ERK and MKK during mitosis using antibodies that specifically recognize active phosphorylated forms of these enzymes. We report the novel finding that ERK and MKK are activated early in prophase before nuclear envelope breakdown, then becoming localized at spindle poles later in prophase. Localization of ERK and MKK is not entirely overlapping, in that active MKK is excluded from condensed chromosomes, whereas active ERK associates with kinetochores and within the chromosomal periphery of condensed chromosomes. This result suggests that ERK phosphorylation by MKK may be involved in chromosomal targeting. A functional role for ERK as a sensor or effector for mitotic progression is suggested by correlations between the appearance and disappearance of active ERK at kinetochores, with those of the antigen(s) recognized by the 3F3/2 monoclonal antibody. Previous studies have shown that this antibody recognizes kinetochore phosphoantigens that respond to spindle fiber attachment (Gorbsky and Ricketts, 1993; Nicklas et al., 1995), and that microinjection of 3F3/2 antibodies delays anaphase entry, suggesting that the phosphoantigen is involved in regulating metaphase-to-anaphase transition (Campbell and Gorbsky, 1995). Our studies with isolated chromosomes indicate that the 3F3/2 epitope is directly or indirectly phosphorylated in response to ERK, suggesting novel roles for ERK in somatic cell mitosis. Materials and Methods Antibodies, Enzyme Purification, and Immunoblotting Affinity-purified rabbit polyclonal antibody to diphosphorylated ERK2 (anti-ACTIVE MAPK) was purchased from (Madison, WI), and mouse monoclonal antibody to diphosphorylated ERK2 was a generous gift of Dr. Rony Seger (Yung et al., 1997). In experiments performed to determine the specificity of the anti-ACTIVE MAPK antibody, wild-type or mutant (His)6-rat ERK2 (Robbins et al., 1993) were expressed in bacteria, purified by Ni+2-nitrilotriacetic acid (NTA) metal affinity chromatography (QIAGEN Inc., Valencia, CA), and phosphorylated for 10 min at 30C with constitutively active mutant MKK1 (G1C: N4/S218E/S222D; Mansour et al., 1996), which was expressed in bacteria and purified as described (Mansour et al., 1994). Reactions contained 1 g ERK2, 1 g MKK1, 0.1 mM ATP, 10 mM MgCl2, 20 mM Hepes, pH 7.4, and 1 mM dithiothreitol in 25 l. Alternatively, whole cell lysates were prepared from NIH 3T3 cells starved in DMEM, 0% FBS overnight, and then treated for 5 min with 10% serum and 0.1 M PMA. Proteins were separated by SDS-PAGE, transferred to Immobilon (Life Science, Inc., Arlington Heights, IL). Immunoblots were also probed using a rabbit polyclonal antibody recognizing the COOH terminus of ERK2 (C-14; (Beverly, MA). To test its specificity, wild-type (His)6-human MKK1 was expressed in bacteria, purified by Ni+2-NTA affinity chromatography (Mansour et al., 1996), proteolyzed with enterokinase (Invitrogen Corp., Carlsbad, CA), and phosphorylated with a constitutively active mutant of MEK kinase ([His]6-MEKKC; Khokhlatchev et al., 1996) for 3 Capromorelin h with 4 mM ATP, 15 mM MgCl2. This test resulted in monophosphorylated and diphosphorylated forms of MKK1 that were subsequently resolved by FPLC using a Mono Q HR5/5 column equilibrated in 20 mM Tris, pH 8, 10% (vol/vol) glycerol, 1 mM dithiothreitol, and were eluted with a linear sodium chloride.