Tuning microtubule dynamics to enhance cancer therapy by modulating FER-mediated CRMP2 phosphorylation

Though used widely in cancer therapy, paclitaxel only elicits a response in a fraction of patients. A strong determinant of paclitaxel tumor response is the state of microtubule dynamic instability. However, whether the manipulation of this physiological process can be controlled to enhance paclitaxel response has not been tested. Here, we show a previously unrecognized role of the microtubule-associated protein CRMP2 in inducing microtubule bundling through its carboxy terminus. This activity is significantly decreased when the FER tyrosine kinase phosphorylates CRMP2 at Y479 and Y499. The crystal structures of wild-type CRMP2 and CRMP2-Y479E reveal how mimicking phosphorylation prevents tetramerization of CRMP2. Depletion of FER or reducing its catalytic activity using sub-therapeutic doses of inhibitors increases paclitaxel-induced microtubule stability and cytotoxicity in ovarian cancer cells and in vivo. This work provides a rationale for inhibiting FER-mediated CRMP2 phosphorylation to enhance paclitaxel on-target activity for cancer therapy

Summary of the results.

<p><b>Each panel</b> shows a monomer of FLN with ABD in red, followed by 24 Ig repeats and dimerization domain in blue. <b>Upper panel</b> shows previously known domain pairs in the Rod 2 region, FLN 16–17, 18–19 and 20–21 in lemon, purple and mustard, respectively. The domains that share high sequence similarity with pair forming domain 21 are highlighted with a star. <b>Middle panel</b> shows the recently revealed three-domain module in the Rod 1 region (Domain 3 in yellow, 4 in green and 5 in cyan). In addition, the arrangement of the three-domain pairs of the Rod 2 region is depicted. <b>Lower panel</b> shows the new candidates for inter-domain interactions revealed in this study.</p

Logarithmic scattering curves as a function of momentum transfer (s).

<p>(<b>A</b>) Comparison of all the constructs studied. (<b>B</b>) Comparison of compact two-domain fragments with the average (FLNc10-11) and completely extended fragments (FLNc8-9). The curves are displaced on the Y-axis for clarity.</p

Parameters derived from SAXS measurements.

$<p>The values written here are with an approximate error of 0.5 nm.</p><p>Parameters derived from SAXS measurements.</p

Distance distribution profile and dimensionless Kratky plots.

<p>(<b>A</b>) P(r) vs r plot of all the constructs. This is supported by Supporting Information S4 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107457#pone.0107457.s001" target="_blank">File S1</a>. (<b>B</b>) Dimensionless Kratky plots of the four compact, average extended and most extended fragments compared to BSA (folded protein) and Tau (unfolded protein).</p

EOM analyses of all constructs.

<p>(<b>A</b>) R_g distribution profile. (<b>B</b>) D_max distribution profile. Dotted lines represent the distribution of the random pool, and solid lines show the distribution of the selected models. Top three panels are made by nudging the curves for clarity.</p

Parameters derived from EOM analysis.

<p>Parameters derived from EOM analysis.</p

<i>Ab-initio</i> envelopes and rigid body models of selected two-domain fragments.

<p>The most compact two-domain fragments are domains 4–5 (<b>B</b>), 3–4 (<b>A</b>) and 14–15 (<b>D</b>); the fragment of domains 11–12 is intermediate (<b>C</b>). Domains 8–9 form the most extended two-domain fragment (<b>E</b>) and domains 10–11 is the average two-domain fragment (<b>F</b>). In the lower panels, the <i>ab-initio</i> and rigid body models are superimposed and their fits are expressed in terms of NSD. The fits of the rigid body models with the experimental scattering are given in the upper panels and the goodness of the fits is expressed in terms of X². Please see Supporting Information S5 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107457#pone.0107457.s001" target="_blank">File S1</a> for models of the rest of the fragments.</p