KXGS sites S319, S350, and S382 are major targets for in vitro phosphorylation of MAP2c by PKA. (A) Recombinant MAP2c was phosphorylated by using purified PKA in vitro as described in MATERIALS AND METHODS. Phosphorylation reached saturation within 30 min, attaining a maximum stoichiometry of 3 mol phosphate/mol MAP2c. (B) wt and mutant MAP2c were phosphorylated in the presence of PKA for 30 s and separated by SDS-PAGE (top). Excised bands were subjected to 2-D phosphopeptide mapping (bottom) by using electrophoresis in the horizontal direction, followed by ascending chromatography in the vertical direction. Mutation of S350 resulted in substantial reduction of total ³²P incorporation. Mutation of S319, S350, and S382 eliminated major spots from the 2-D phosphopeptide map (arrowheads).

Effect of PKA on colocalization of MAP2c with microtubules in living HeLa cells. S350 is a major target of PKA in vivo. (A) Expression of MAP2 in HeLa cells causes remodeling of endogenous microtubule structure. Cultured HeLa cells were transiently transfected with constructs encoding wt or mutant GFP-MAP2c and incubated in the absence or presence of 20 μM forskolin for 10 min. In each row are shown two representative images of cells from each experimental group (designated at left). Bars, 10 μm. Top, HeLa cells expressing wt GFP-MAP2c that were incubated with vehicle alone contain large arrays of stable microtubules that accumulate inperipheral bundles highly enriched in GFP-MAP2c. Row 2, forskolin treatment resulted in reduced colocalization of MAP2c with microtubules and increased cytoplasmic fluorescence. Rows 3 and 4, mutation of serine 350 to alanine (S350A) eliminated forskolin-dependent changes in GFP-MAP2c localization. (B) Quantitative image analysis of control and forskolin-stimulated HeLa cells expressing MAP2c and S350A MAP2c. Mean and SD of pixel values were determined for each image and used to express the variation in fluorescence intensity within the cell as described in MATERIALS AND METHODS. Forskolin caused a substantial increase in the localization of wt GFP-MAP2c fluorescence in the cytoplasm, reflected in significantly lower pixel variation (P < 0.0001, Student's t test, unpaired; n > 50 for each group). This effect was eliminated in the S350A GFP-MAP2c (P > 0.05, Student's t test, unpaired, n > 50 for each group). Bars represent mean ± SE.

PKA activation promotes MAP2 association with actin. HeLa cells expressing wt GFP-MAP2c (green) were incubated with vehicle alone (A) or with 20 μM rolipram plus 10 μM forskolin for 10 h (B) as described in MATERIALS AND METHODS. Cells were fixed and immunolabeled for actin (red). Stacks of images were collected and 3-D reconstructions were created as described in MATERIALS AND METHODS. Shown are maximal projections of a series of single plane (x-y) images collected at 0.2-μm intervals in the z-dimension. The boxed regions are shown in magnified views for the actin (red), GFP-MAP2c (green), and merged images as indicated. Bars, 10 μm. (A) In control, unstimulated cells MAP2c is highly colocalized with microtubules and absent from peripheral regions enriched in actin. MAP2 is absent from actin structures within the ruffle. Video supplement OzerFig 3A.mov shows a revolving 3-D reconstruction of the merged image. (B) PKA activation promotes MAP2c–actin colocalization. Neither protein fills the cytoplasmic volume; however, GFP-MAP2c is partially colocalized within specific structures where actin is enriched (arrowheads). Video supplement OzerFig 3B.mov shows a revolving 3-D reconstruction of the merged image.

Mutation of KXGS serines to glutamic acid disrupts MAP2–microtubule association in living cells. Transfected HeLa cells expressing wt or mutant GFP-MAP2c were partitioned into soluble (S) and cytoskeletal pellet (P) fractions as described in MATERIALS AND METHODS. Quantitative immunoblot analysis showed that, relative to wt, each mutant form of MAP2c (M) was enriched at least 1.5-fold in the soluble fraction. Consistent with earlier work (Minotti et al., 1991), ∼40% of cellular tubulin (T) was observed in the soluble fraction in all cases. All mutants were significantly different from wt (P < 0.01) and none were significantly different from each other (P > 0.05, one-way ANOVA followed by Tukey's multiple comparison test, n = 5). Bars represent mean ± SE.

Single KXGS mutations reduce MAP2c–microtubule interaction. Living HeLa cells transfected to express either wt or mutant GFP-MAP2c were imaged by confocal microscopy as described in MATERIALS AND METHODS. Bar, 10 μm. (A) wt GFP-MAP2c remodels endogenous microtubules into hyperstabilized bundles. (B) S319E mutation resulted in reduced remodeling of endogenous microtubules, although MAP2c–microtubule binding appeared to be retained. (C) S350E mutation resulted in increased cytoplasmic MAP2c and the appearance of negatively outlined organelles. (D) S382E mutation resulted in increased cytoplasmic MAP2c and the appearance of negatively outlined organelles. (E) Quantitative image analysis of the effects of mutations at S319, S350, and S382. Mean and SD were determined for each image as described in MATERIALS AND METHODS and used to express the variation in pixel intensity within the cell. All mutations induced a substantial change in the localization of GFP, reflected in significantly lower pixel variation (P < 0.001, one-way ANOVA followed by Tukey's multiple comparison test, n ≥ 50/group). The S319E mutant was significantly different from the S350E and S382 mutants (P < 0.001.); however, the S350E mutant was not significantly different from the S382E mutant (P > 0.05). Bars represent mean ± SE.

Multiple KXGS mutations alter subcellular localization. Living HeLa cells transfected to express either wt or mutant GFP-MAP2c were imaged by confocal microscopy as described in MATERIALS AND METHODS. Bar, 10 μm. (A) wt GFP-MAP2c remodels endogenous microtubules into hyperstabilized bundles and is excluded from the cellular periphery. (B–D) S319E/S382E (B), S350E/S382E (C), and S319E/S350E/S382E (D) mutations all resulted in increased cytoplasmic MAP2c and the appearance of negatively outlined organelles to the same degree as the S350E or S382E single mutations. Multiple mutations promoted localization to peripheral ruffles (arrowheads). (E) Quantitative image analysis of the effects of multiple mutations at S319, S350, and S382. Mean and SD were determined for each image as described in MATERIALS AND METHODS and used to express the variation in pixel intensity within the cell. All mutations induced a substantial change in the localization of GFP, reflected in significantly lower pixel variation (P < 0.001, one-way ANOVA followed by Tukey's multiple comparison test, n ≥ 50/group). The multiple mutants were not significantly different from each other or from the single mutants S350E and S382E shown in Figure 5 (P > 0.05). Bars represent mean ± SE.

Mutant MAP2c localizes to motile peripheral ruffles. Living HeLa cells transfected to express either wt or mutant GFP-MAP2c were imaged by confocal microscopy as described in MATERIALS AND METHODS. Bar, 10 μm. (A) wt GFP-MAP2c is highly colocalized with microtubules and absent from the cellular periphery. (B) S350E/S382E GFP-MAP2c promotes pseudopod-like structures, which were rarely observed in untransfected HeLa cells. (C and D) Localization to ruffles is not dependent on expression level. Adjacent cells with disparate (C) or similar (D) expression levels of S350E/S382E GFP-MAP2c contain MAP2 in peripheral ruffles to a similar degree. (E) Simultaneous time-lapse differential interference contrast (right) and fluorescence (left) imaging revealed that peripheral regions enriched in S350E/S382E GFP-MAP2c (arrowheads) exhibited a high degree of motility suggestive of membrane ruffling. Shown are single frame captures from two independent time-lapse series. Bar, 10 μm.

S350E/S382E mutant MAP2c is not restricted to microtubule-containing domains. HeLa cells expressing wt and S350E/S382E GFP-MAP2c were fixed and immunolabeled for tubulin. wt MAP2c remodeled endogenous microtubules into bundles at the periphery of the cell and MAP2c was restricted to domains of the cell also enriched in tubulin. In cells expressing S350E/S382E GFP-MAP2c, endogenous microtubules appeared similar to those in nontransfected HeLa cells because they rarely exhibited microtubule bundles and most microtubules appeared to radiate centrifugally. In addition, mutant MAP2c was enriched in peripheral structures that did not contain microtubules (arrowheads). Bar, 10 μm.

S350E/S382E mutant GFP-MAP2c is colocalized with actin in peripheral ruffles. HeLa cells expressing wt and S350E/S382E GFP-MAP2c were fixed and immunolabeled for actin. wt MAP2c was absent from peripheral areas of the cell and did not significantly colocalize with actin. In contrast, S350E/S382E MAP2c was enriched in peripheral ruffles that were also highly enriched in actin (arrowheads). Bar, 10 μm.

Specific association between mutant MAP2c and the actin cytoskeleton. (A) Unfixed HeLa cells expressing GFP, GFP-MAP2c, or S350E/S382E GFP-MAP2c were imaged before (untreated) and after extraction (extracted) with a detergent-containing buffer as described in MATERIALS AND METHODS. Virtually all soluble cytoplasmic GFP was removed by this protocol (top). The subcellular distribution and fluorescence intensity of wt GFP-MAP2c was largely unchanged by this procedure (middle). In contrast, the majority of S350E/S382E mutant GFP-MAP2c was extracted (bottom). Labeling was retained in peripheral membrane areas (arrowheads) and along filamentous structures (note thatacquisition parameters were adjusted for display purposes). Bar, 10 μm. (B) After detergent extraction, S319E/S382E GFP-MAP2c was retained in structures resembling actin-based stress fibers, which were prevalent near the bottom of the cell, closest to the glass coverslip. Left, mutant GFP-MAP2c imaged in an unfixed cell after extraction. Right, actin filaments labeled with rhodamine phalloidin in fixed HeLa cells. Bars, 10 μm.