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Association of mitogen-activated protein kinases with microtubules in mouse macrophages

Taxol, a microtubule-binding diterpene, mimics many effects of lipopolysaccharide (LPS) on mouse macrophages. The LPS-mimetic effects of taxol appear to be under the same genetic control as responses to LPS itself. Thus we have postulated a role for microtubule-associated proteins (MAP) in the response of macrophages to LPS. Stimulation of macrophages by LPS quickly induces the activation of mitogen-activated protein kinases (MAPK). MAPK are generally considered cytosolic enzymes. Herein we report that much of the LPS-activatable pool of MAPK in primary mouse peritoneal macrophages is microtubule associated. By immunofluorescence, MAPK were localized to colchicine- and nocodazole- disruptible filaments. From both mouse brain and RAW 264.7 macrophages, MAPK could be coisolated with polymerized tubulin. Fractionation of primary macrophages into cytosol-, microfilament-, microtubule-, and intermediated filament-rich extracts revealed that approximately 10% of MAPK but none of MAPK kinase (MEK1A and MEK2) was microtubule bound. Exposure of macrophages to LPS did not change the proportion of MAPK bound to microtubules, but preferentially activated the microtubule- associated pool. These findings confirm the prediction that LPS activates a kinase bound to microtubules. Together with LPS-mimetic actions of taxol and the shared genetic control of responses to LPS and taxol, these results support the hypothesis that a major LPS-signaling pathway in mouse macrophages may involve activation of one or more microtubule-associated kinases.


Summary
Taxol, a microtubule-binding diterpene, mimics many effects of lipopolysaccharide (LPS) on mouse macrophages. The LPS-mimetic effects of taxol appear to be under the same genetic control as responses to LPS itself. Thus we have postulated a role for microtubule-associated proteins (MAP) in the response of macrophages to LPS. Stimulation of macrophages by LPS quickly induces the activation of mitogen-activated protein kinases (MAPK). MAPK are generally considered cytosolic enzymes. Herein we report that much of the LPS-activatable pool of MAPK in primary mouse peritoneal macrophages is microtubule associated. By immunofluorescence, MAPK were localized to colchicine-and nocodazole-disruptible filaments. From both mouse brain and RAW 264.7 macrophages, MAPK could be coisolated with polymerized tubulin. Fractionation of primary macrophages into cytosol-, microfilament-, microtubule-, and intermediate filament-rich extracts revealed that "~10% of MAPK but none ofMAPK kinase (MEK1 and MEK2) was microtubule bound. Exposure of macrophages to LPS did not change the proportion of MAPK bound to microtubules, but preferentially activated the microtubule-associated pool. These findings confirm the prediction that LPS activates a kinase bound to microtubules. Together with LPS-mimetic actions of taxol and the shared genetic control of responses to LPS and taxol, these results support the hypothesis that a major LPS-signaling pathway in mouse macrophages may involve activation of one or more microtubuleassociated kinases.
M itogen-activated protein kinases (MAPK), originally termed microtubule-associated protein (MAP) 2 kinase and later also called extracellular signal-regulated kinases (ERK), are rapidly activated in response to various extracellular stimuli in many cell types via a cascade (1-4) that eventuates in phosphorylation of the enzymes on both tyrosine and threonine residues (5). Substrates for MAPK are found in the nucleus, plasma membrane, cytosol, and cytoskeleton (4, 6), but it is not entirely clear which are physiologic and how MAPK, generally considered cytosolic, encounter them. Activation of MAPK by bacterial LPS (7,8) is one of the most rapid knowneffects of LPS on macrophages (9) and may be required for the LPS-triggered release ofeicosanoids (7) and cytokines (10,11).
Taxol, a microtubule-binding diterpene, exerts cell cycle-independent effects on macrophages strikingly similar to those of LPS (12). These include downregulation of TNF receptors (12), activation of MAPK (9,13), mobilization of nuclear factor (NF)-gB (14), and induction of TNF (12) and other early genes (13,15). These responses are absent in macrophages from C3H/HeJ mice (12)(13)(14)(15), which bear a defective allele of the Lps gene on chromosome 4. LPS-mimetic responsiveness to taxol cosegregated with the normal Lps allele in nine recombinant inbred strains (12). Inactive LPS analogues blocked taxol-induced protein tyrosine phosphorylation and expression of proinflammatory genes (16). These observations support the hypothesis that LPS and taxol share a common target in a signaling pathway controlled by the Lps gene (12). Although LPS and taxol may activate such a target directly from the cell surface, both enter cells rapidly (17,18) and bind specifically to [3-tubulin in a cell-free system (19)(20)(21). Thus, whether they are activated directly or indirectly, the common target of LPS and taxol may be MAP. The present report establishes that MAPK itself is one such LPS-and taxol-activated MAP.
Isolation of MAP. MAP were prepared from 10 mouse brains by the procedure of Vallee (23). In brief, tubulin in the homogenate supemate (180,000 g) was polymerized at 37~ by the addition oftaxol and GTP, and the resulting microtubules were centrifuged through sucrose. MAP were stripped fi'om the microtubules with 0.4 M NaCI and recovered in the 30,000 g supemate. By silver-stained SDS-PAGE, the MAP preparation contained one major species migrating at 220 kD and one minor species at 55 kD. These were specifically recognized by antibodies against MAP-2 and tubulin, respectively (not shown). The 30,000 g sediment (used below) contained only tubulin and no MAP by silverstained SDS-PAGE and immunoblot. Similarly, ,,o101~ R A W 264.7 cells were homogenized in 10 mM EDTA, 1 mM MgSO4, pH 6.6, with 1 mM PMSF and 5 ~g/ml each pepstatin A, leupeptin, aprotinin, and chymostatin, and sequentially centrifuged at 4~ (170, 30,000, and 180,000 g) to remove unbroken cells, nuclei, and microsomes. MAP were then isolated as for brain except that 0.6 mg purified brain tubulin was added as scaffold.
Differential Cytoskeletal Extractions. Based on the procedure of van Bergen en I-Ienegouwen et al. (24), macrophages were washed in cytoskeleton-stabilizing buffer (CSK) containing 10 mM Pipes, pH 6.8, 250 mM sucrose, 3 mM MgC12, 150 mM KC1, 1 mM EGTA, and 1 mM PMSF and lysed at 37~ in CSK buffer containing 0.15% Triton X-100 (lysis buffer) for 5 min. Supemate $1 (14,000 g, 10 rain, room temperature) was considered the cytosolic fraction. Pellet1 was washed three times in lysis buffer at 37~ Microtubules were depolymerized by chilling the samples to 4~ in the same buffer and collected as $2 (14,000 g, 10 rain) followed by two washes with cold lysis buffer. Pellet2 containing actin-based microfilaments was solubilized with 0.6 M KC1 in CSK buffer in the presence of DNase (0.2 mg/ml) and MgC12 (10 mM), followed by centrifugation (14,000 g, 20 rain), generating $3. The remaining pelle h containing intermediate filaments was dissolved in Laemmli's sample buffer with SDS. For kinase assay, extracts were prepared in the presence of 1 mM each of sodium vanadate, sodium pyrophosphate, and sodium fluoride.
Immunoblot. Samples separated by SDS-PAGE were transferred to a nitrocellulose membrane (Schleicher & Schuell, Inc., Keene, NH) in an ice-water bath. Membranes were blocked in 20 mM Tris, 137 mM NaCI, pH 7.4, plus 0.1% Triton X-100 (TBST) containing 10% FBS or 5% dry milk at 4~ overnight, incubated with first antibody for 1 h at room temperature, washed three times with TBST, and incubated with horseradish peroxi-dase-conjugated secondary antibody (1:10,000 in TBST) for 45 min at room temperature. After extensive washing with TBST, immunoblots were developed using an enhanced chemiluminescence kit (Amersham Corp., Arhngton Heights, IL).

Localization of M A P K to Microtubules in Intact, Primary
Macrophages. Anti-MAPK mAb-stained filamentous structures were unaltered by prior exposure to LPS (Fig. 1 A) but were dissassembled by preincubation (2 h) with the microtubule-disrupting agents colchicine (10 IzM) (Fig. 1 B) or nocodazole (10 ~M) (not shown). The pattern o f MAPK distribution matched that observed with antitubttlin mAb, except that anti-MAPK did not decorate microtubuleorganizing centers (Fig. 1 C). Entirely different staining patterns resulted with antivimentin (marking intermediate filaments, Fig. 1 D) or rhodamine-conjugated phaUoidin (revealing actin-rich microfilaments, Fig. 1 E). In contrast to the situation with growth factor-treated fibroblasts (26,27), LPS did not cause MAPK to translocate to the macrophage nucleus.
MAPK Are among the MAP Isolated fiom Mouse Brain and RA W 264. 7 Cells. MAP are operationally defined by their cosedimentation with polymerized microtubules through a sucrose gradient (28). MAP from mouse brain and from the macrophage-hke cell line RAW 264.7 prepared in this way contained 42-and 44-kD MAPK (Fig. 2), well separated from actin (here migrating at <40 kD) and tubulin (55 kD), the identity of the latter proteins being confirmed by immunoblot. The several species binding anti-MAP-2 ( Fig.  2) may correspond to known MAP-2 isoforms (29).
A Portion of MAPK Specifically Cofractionates with Microtubules in Primary Macrophages. The next experiments used a sequential extraction scheme (24) that yields fractions enriched in each of three major classes of cytoskeletal structures. About 75% of total cellular protein was recovered as cytosolic ($1). After three washes of pellet1, microtubule proteins (-'~7%) were recovered by depolymerization in the cold ($2). Actin filaments ("~8%) were removed by high salt extraction ($3), and a portion of intermediate filaments remained associated with the final pellet and were extracted in SDS ($4). Each fraction was immunoblotted with antibodies against compamilent markers (Fig. 3): MAPK kinase (MEK1 and MEK2) for cytosol, tubulin for microtubules, actin for microfilaments, and vimentin for intermediate illaments. MEK was detected only in the cytosolic fraction, in contrast to observations in NIH/3T3 cells mentioned in abstract form (30). Tubulin was present in both cytosohc and microtubule fractions, consistent with the normal equihbrium between polymerized and depolymerized microtubules. Similarly, as expected, actin was both soluble and microfilament associated. Vimentin was insoluble in Triton X-100 and was only detected in the high salt extract and its SDS-soluble residue (Fig. 3). MAPK was absent in the microfilament-and intermediate filament rich fractions (Fig. 4 A, lanes 3 and 4) and present in the cytosolic and microtubule-rich fractions (Fig.  4 A, lanes 1 and 2). As already noted, MEK was present in cytosol but not in microtubule-rich fractions, arguing against nonspecific contamination of the latter with the former. Moreover, supernates of three washes of pelleh were analyzed to exclude the possibility that MAPK from $1 may have associated nonspecifically with pellet1 and thus persisted in S 2. No MAPK were detected in the second or third of these washes. Thus, in primary macrophages, a portion of MAPK is specifically associated with the microtubule network, but not with any other major cytoskeletal fraction. Densitometric comparison of dilutions of these fractions indicated that the microtubule-associated portion of MAPK averaged 10% of the total (8, 9, and 14% in three experiments) (Fig. 4 B).
LPS Preferentially Aaivates Microtubule-Associated MAPK. Macrophages were incubated with 0 or 100 ng/ml of LPS for 15 min, 2 h, or 18 h and subjected to sequential extraction as above. As seen in intact macrophages by immunofluorescence, LPS caused no detectable redistribution of MAPK in fractionated macrophages (Fig. 5 A). However, LPS (100 ng/ml, 15 min) enhanced the enzymatic activity of MAPK more markedly in the microtubule-associated fraction (3.7 + 1.1-fold, six experiments) than in the cytosohc fraction (1.6 + 0.3-fold) (P <0.015, Student's t test; Fig. 5 B).

Discussion
Several parallel MAPK cascades regulate growth hormone-, cytokine-and stress-stimulated responses in diverse cell types (4)(5)(6)(31)(32)(33)(34)(35)(36). The pleiotropic nature of MAPK action is evident from the complex array of substrates localized in different cellular compartments and executing diverse functions (31,(37)(38)(39)(40)(41)(42)(43). The postulate that MAPK must be, at least transiently, in direct contact with their physiological substrates has led to refinement of the initial description of MAPK as strictly cytosolic enzymes. In fibroblasts, mitogenic signals induce translocation of MAPK to nuclei and plasma membrane (26,27). MAPK were found in dendritic microtubules of rat brain cells (44), in microtubule-organizing centers in mouse oocytes during meiotic maturation (45), and in the microtubules of cycling mouse fibroblasts (46). To our knowledge, this report is the first to document a microtubule localization o f M A P K in noncycling cells outside the nervous system, and to relate this association to responses to a microbial product.
Many MAPs, including tau, MAP=l, MAP-2, MAP=5, and caldesmon, can serve as facile substrates o f M A P K (4,47,48). Microtubule=associated M A P K may be the physio= logically relevant pool acting on MAP substrates. Phosphorylation o f MAP regulates microtubule polymerization (49). l~eorganization ofmicrotubules is an important aspect o f cell remodeling in such diverse situations as dendrite for= mation, mitosis, cell spreading, and migration.
The discovery o f LPS-mimetic effects o f taxol on macrophages from C 3 H / H e N (LPS-normoresponsive), but not C 3 H / H e J (LPS-hyporesponsive) mice led to the hypothe= sis that these two agents might share a c o m m o n target (12). LPS=mimetic effects of taxol, however, are cell cycle inde= pendent (12,16). So far no other intraceUular binding site for taxol besides polymerized tubulin has been identified. Thus, binding o f taxol to microtubules may evoke two dis- We wish to thank Dr. Carl Nathan for his support and critical reading of the manuscript. This work was supported by National Institutes of Health grant AI.30165.