Raf-1 is activated during mitosis.

The Raf-1 serine/threonine kinase transmits proliferative and developmental signals to downstream effectors in the mitogen-activated protein kinase cascade. Studies to date have concentrated on its role in growth factor-mediated activation of the cell cycle. We show here that Raf-1 kinase activity is also highly stimulated during mitosis, with an attendant distinctive electrophoretic mobility shift due to hyperphosphorylation. These results suggest that Raf-1 may play a role in traversal of at least two distinct phases of the cell cycle.

The Raf-1 serine/threonine kinase transmits proliferative and developmental signals to downstream effectors in the mitogen-activated protein kinase cascade. Studies to date have concentrated on its role in growth factor-mediated activation of the cell cycle. We show here that Raf-1 kinase activity is also highly stimulated during mitosis, with an attendant distinctive electrophoretic mobility shift due to hyperphosphorylation. These results suggest that Raf-1 may play a role in traversal of at least two distinct phases of the cell cycle.
The Raf-1 kinase is an integral part of a cascade that transmits mitogenic and developmental signals from activated growth factor (GF) 1 receptor tyrosine kinases to the mitogenactivated protein kinases (MAPKs) known as extracellular signal-regulated protein kinases (ERKs) (1,2). Activation of Raf-1 following GF stimulation involves Raf-1/Ras interaction at the plasma membrane (2) and is associated with Raf-1 hyperphosphorylation and retarded mobility in SDS-PAGE (3). Activated Raf-1 phosphorylates and activates the MAPK or ERK kinases (MEKs), dual specificity kinases that in turn phosphorylate and activate the ERKs (1,4). The ERKs phosphorylate a number of cytoplasmic and nuclear targets, resulting in changes in transcription and translation (4).
Studies on proto-oncoproteins have focused on their roles in the G 0 /G 1 transition or in traversal of G 1 . A number of protooncoproteins, notably c-Src, c-Abl, and c-Mos, have previously been shown to undergo hyperphosphorylation and modulation of activity in mitosis and/or M-phase (5)(6)(7). In this study we present evidence that Raf-1 becomes activated and hyperphosphorylated in mitosis.
Retroviral Infection-NIH 3T3 cells were infected as described (10) with the retroviral construct pLJ/Raf (a gift from D. Morrison) or the control construct pLJ. Infected cells were grown in mass culture under G418 selection (400 g/ml) for 2 weeks prior to experiments being performed.

RESULTS AND DISCUSSION
We have observed that Raf-1 immunoprecipitated from nocodazole-arrested mitotic NIH 3T3 cells migrates more slowly (74 -78 kDa) in SDS-PAGE than Raf-1 from unsynchronized growing cells (72-74 kDa; Fig. 1, lanes 1 and 2). Following potato-acid phosphatase treatment this mobility retardation was virtually eliminated, demonstrating that it stems from hyperphosphorylation (lanes 3 and 4). Pretreatment of potatoacid phosphatase with a mixture of phosphatase inhibitors (sodium fluoride and sodium vanadate) inhibited this effect (data not shown). To further study alterations of Raf-1 in mitosis we used the retroviral expression vector pLJ/Raf-1 to generate NIH 3T3 cell lines (designated NIH(pLJ/Raf-1)) that stably overexpress human Raf-1. NIH(pLJ) cell lines that were infected with the empty vector pLJ were used as controls. Direct comparisons with both the endogenous and overexpressed Raf-1 showed that the mitotic reduction in electrophoretic mobility was similar to that observed following 5 min of stimulation with platelet-derived growth factor (PDGF) ( Fig.  2A, cf. lanes 3 and 2 and lanes 7 and 6). Multiple mobilityretarded Raf-1 bands were observed in both PDGF-treated and mitotic cells, but more highly retarded bands were more abundant in mitotic Raf-1. Raf-1 retardation increased following longer treatment with PDGF but never to the extent observed with Raf-1 from mitotic cells (data not shown).
NIH(pLJ/Raf-1) mitotic and unsynchronized cell cultures were metabolically labeled with inorganic 32 PO 4 , and immunoprecipitated Raf-1 was analyzed by phosphoamino acid analysis (Fig. 2B). Raf-1 isolated from both mitotic and unsynchronized cells was radiolabeled predominantly on serine residues, but low levels of threonine phosphorylation were also detected. This is similar to results observed following PDGF stimulation of NIH 3T3 cells (12).
To determine the duration of Raf-1 hyperphosphorylation in mitosis, NIH(pLJ/Raf-1) cells were arrested in mitosis using nocodazole, replated in drug-free medum, and harvested at different intervals (Fig. 2C). The mitotic mobility retardation of Raf-1 was partially lost by 1 h after replating (lane 4) and was negligible by 2 h (lane 5). Since the cells undergo cytokinesis 30 -45 min after release from nocodazole arrest (13), it appears that Raf-1 hyperphosphorylation extends into the early G 1 phase of the cell cycle. This contrasts with the case of c-Src, where Cdc2-catalyzed hyperphosphorylations disappear within 30 min after replating (data not shown and Ref. 13). Similar results for Raf-1 were obtained using HeLa cells. 2 Two different assays were used to determine if Raf-1 kinase activity is stimulated during mitosis: 1) an autokinase assay and 2) phosphorylation of a relevant physiological substrate, i.e. purified recombinant MEK-1. Autophosphorylation was assayed using Raf-1 immunoprecipitated from quiescent (serumstarved), PDGF-stimulated, mitotic, and unsynchronized NIH(pLJ/Raf-1) cells (Fig. 3A). As anticipated (3), Raf-1 immunoprecipitated from quiescent (lane 1) or unsynchronized (lane 4) cultures exhibited negligible autokinase activity. Importantly, autokinase activity was stimulated in mitotic cells to a level comparable with that observed following PDGF treatment (cf. lanes 3 and 2). Phosphoamino acid analysis revealed that both PDGF-stimulated and mitotic Raf-1 were autophosphorylated on both serine and threonine (Fig. 3B). The MEK kinase activity of Raf-1 was also stimulated in mitotic cells (Fig. 3C). The activity of Raf-1 from mitotic cells was 10 ϫ/Ϭ 2 (n ϭ 4) times higher than the activity of Raf-1 from unsynchronized cells. The level of mitotic activity was very similar to that observed following PDGF stimulation (data not shown). Definitive investigation of the role of the mitotic hyperphosphorylation of Raf-1 in its activation must await mapping and mutation of the mitotic phosphorylation sites.
To ensure that Raf-1 hyperphosphorylation and activation were not drug-induced artifacts, we performed similar experiments using mitotic cells collected by mechanical shake-off alone (Fig. 3D). Western blots of immunoprecipitates revealed that Raf-1 mobility was retarded, although to a lesser extent than that of Raf-1 isolated from nocodazole-treated cells (Fig.  3D, lower panel). Similarly, Raf-1 kinase activity was stimulated about 3-fold in mitotic cells isolated by shake-off alone (Fig. 3D, upper panel, lane 6) compared with Raf-1 in immunoprecipitates from the adherent monolayer remaining following shake-off (lane 5) or from unsynchronized cells (lane 7). This stimulation was somewhat less than that observed in nocodazole-treated cultures (lane 8), but for reasons discussed elsewhere (13) we believe that this merely reflects heterogeneity of the shake-off population and conclude that the observed mitotic hyperphosphorylation and stimulation of Raf-1 kinase activity are authentic.
We cannot exclude the possibility that the observed enhancement of Raf-1 kinase activity comes from the mitosis-specific association or activation of another serine/threonine kinase that coprecipitates with Raf-1. We believe this is unlikely since 1) MEK-1 appears to be a target for a very restricted set of kinases (1) and 2) the lysis buffer contains 0.1% SDS and 0.5% sodium deoxycholate, which should disassociate any such activity from Raf-1 prior to Raf-1 immunoprecipitation (8). In any case, if such an association/activation occurs, it is specific to mitosis and would therefore itself be of great interest.
Raf-1 activity is already known to participate in the G 0 /G 1 transition. The data presented here suggest that it may also function in the transition into or passage through mitosis. A reduction in the electrophoretic mobility of Raf-1 in mitotic murine fibroblasts has been previously reported, but no changes in Raf-1 activity were described (14). In future experiments it will be important to determine 1) if Raf-1 is first activated before or during mitosis, 2) if the mechanism of its mitotic activation is related to the mechanism employed following GF stimulation (i.e. via Ras (2)), and 3) its mitotic targets. Interestingly, there is already evidence indicating that a Raf-1-mediated pathway(s) participates in entry into meiosis. For example, activated GF receptor tyrosine kinases stimulate Xenopus meiotic maturation in a Raf-1-dependent manner (15), and progesterone-initiated maturation is partially Raf-1-dependent (15,16). Furthermore, ERK1 (which lies downstream from Raf-1) regulates a meiotic checkpoint 2 S. Taylor and D. Shalloway, unpublished observations.

FIG. 2. Characterization and duration of Raf-1 mitotic shift.
A, comparison of the mobility retardations of Raf-1 isolated from PDGFstimulated and mitotic cells. Lysates were prepared from NIH(pLJ) and NIH(pLJ/Raf-1) cells that had been serum-starved for 24 h (Ϫ), serumstarved followed by 5 min, 20 ng/ml PDGF treatment (ϩ), that had been arrested in mitosis by nocodazole-treatment (M), or that were unsynchronized (U). Samples containing Raf-1 immunoprecipitates from 250 g of total cell protein were separated by 8% SDS-PAGE, Western blotted, and probed with Raf-1 antibody (upper panel). A weaker exposure of lanes 5-8 is included (lower panel). The apparent reduction of the Raf-1 signal in PDGF-stimulated and mitotic NIH(pLJ) cells (lanes 2 and 3) and in PDGF-stimulated NIH (pLJ/Raf-1) cells (lanes 6) was not reproducible. B, Raf-1 is phosphorylated predominantly on serine residues. Mitotic (M) NIH(pLJ/Raf-1) cells were metabolically labeled in reduced phosphate medium by arresting with nocodazole and then adding 2.0 mCi of 32 PO 4 and incubating (in the continued presence of nocodazole) for 2 more h. Unsynchronized (U) cells were similarly labeled except without nocodazole. Raf-1 was immunoprecipitated and Western blotted as in panel A. Following autoradiography 32 P-labeled Raf-1-containing bands were excised and subjected to phosphoamino acid analysis by partial acid hydrolysis, thin layer chromatography, and autoradiography. The positions of nonradioactive phosphoserine (S), phosphothreonine (T), and phosphotyrosine (Y) standards are indicated. C, time course showing increased Raf-1 electrophoretic mobility following release from mitotic arrest. Nocodazole-arrested mitotic NIH (pLJ/Raf-1) cells were replated in drug-free medium and incubated for the indicated times (lanes 2-7). Lysates were prepared, and 30-g fractions were analyzed by 8% SDS-PAGE, Western blotted, and probed with a polyclonal Raf-1 antibody. Unsynchronized (U) cell lysates (lanes 1 and 8) were used as controls. The two-three distinct Raf-1 bands that are evident in C are preferentially recognized by the polyclonal antibody used in this experiment. A monoclonal antibody that recognizes an unresolved collection of multiple bands was used in all other displayed experiments. Similar kinetics were observed for the postmitotic disappearance of the Raf-1 electrophoretic mobility shift with both antibodies.
FIG. 1. The electrophoretic mobility of Raf-1 is retarded in mitotic NIH 3T3 cells due to hyperphosphorylation. Lysates were harvested from exponentially growing unsynchronized (U) and nocodazole-arrested (M) cells. Nocodazole-arrested cells were prepared as described previously (13). Immunoprecipitated Raf-1 was incubated without (Ϫ) or with (ϩ) potato-acid phosphatase (PAP) as described previously (35), subjected to 8% SDS-PAGE, Western blotted, and probed with Raf-1 antibody. Bands were visualized by chemiluminescence. The broad Raf-1 band actually consists of at least six electrophoretically distinct species. The positions of molecular weight standards (in kDa) are shown.
MEK-1 is the most obvious potential target for Raf-1 during mitosis. Raf-1-catalyzed phosphorylation and activation of MEK-1 could explain the activation of MAPKs that occurs just before or during mitosis in mammalian cells (23,24). However, Raf-1 might phosphorylate and modulate other targets such as IkB or p53 (25,26). The latter possibility is of particular interest because p53 may help regulate the G 2 /M transition (27,28).
The recent finding that Raf-1 phosphorylates and activates Cdc25A, an activator of cyclin-dependent kinases, during cell cycle activation further indicates that it may play a significant role in cell cycle regulation (29).
We do not yet know if the mitotic hyperphosphorylation of Raf-1 is a cause or effect of its activation. It will be important to test the possibility that Src (or other tyrosine kinases) plays a role in the mitotic activation of Raf-1. Microinjection of anti-Ras inhibitory antibodies reverts the transformed phenotypes of v-Src-transformed cells, demonstrating that v-Src acts upstream of the Ras/Raf-1 pathway (30). Moreover, oncogenic Src can activate Raf-1 in insect and mammalian cells (9,31,32). We did not detect increased tyrosine phosphorylation of Raf-1 during mitosis (Fig. 2B). However, it should be noted that enhanced tyrosine phosphorylation is confined to a small fraction of Raf-1 molecules following activation by coexpression with oncogenic Ras and Src in mammalian cells, even though tyrosine phosphorylation has been implicated in Raf-1 activation in this situation (32). In vivo mitotic hyperphosphorylation was observed overwhelmingly at serine (Fig. 2B) while Raf-1 autophosphorylates in vitro at both serine and threonine ( Fig.  3B and Refs. 31 and 33). MAPKs are probably responsible for at least some of the Raf-1 hyperphosphorylation seen in GF-stimulated cells (34) and might therefore also contribute to Raf-1 mitotic hyperphosphorylation in a feedback loop.
The results presented here indicate that Raf-1 must be added to the list of proto-oncoproteins whose activities become modulated in mitosis and suggest that deregulated Raf-1 activity could perturb control mechanisms at multiple points in the cell cycle.