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Abstract
An oncogenic form of RHAMM (receptor for hyaluronan-mediated motility, mouse, amino acids 163-794 termed RHAMM(Delta163)) is a cell surface hyaluronan receptor and mitotic spindle protein that is highly expressed in aggressive human cancers. Its regulation of mitotic spindle integrity is thought to contribute to tumor progression, but the molecular mechanisms underlying this function have not previously been defined. Here, we report that intracellular RHAMM(Delta163) modifies the stability of interphase and mitotic spindle microtubules through ERK1/2 activity. RHAMM(-/-) mouse embryonic fibroblasts exhibit strongly acetylated interphase microtubules, multi-pole mitotic spindles, aberrant chromosome segregation, and inappropriate cytokinesis during mitosis. These defects are rescued by either expression of RHAMM or mutant active MEK1. Mutational analyses show that RHAMM(Delta163) binds to alpha- and beta-tubulin protein via a carboxyl-terminal leucine zipper, but in vitro analyses indicate this interaction does not directly contribute to tubulin polymerization/stability. Co-immunoprecipitation and pulldown assays reveal complexes of RHAMM(Delta163), ERK1/2-MEK1, and alpha- and beta-tubulin and demonstrate direct binding of RHAMM(Delta163) to ERK1 via a D-site motif. In vitro kinase analyses, expression of mutant RHAMM(Delta163) defective in ERK1 binding in mouse embryonic fibroblasts, and blocking MEK1 activity collectively confirm that the effect of RHAMM(Delta163) on interphase and mitotic spindle microtubules is mediated by ERK1/2 activity. Our results suggest a model wherein intracellular RHAMM(Delta163) functions as an adaptor protein to control microtubule polymerization during interphase and mitosis as a result of localizing ERK1/2-MEK1 complexes to their tubulin-associated substrates.
Highlights
The interphase microtubule network is composed of ␣and -tubulin heterodimers that form tubules, which are highly dynamic structures participating in cell morphology/ polarity, signaling, migration, proliferation, and protein trafficking [1,2,3]
We were prompted to investigate whether or not intracellular RHAMM protein controls interphase and mitotic microtubule stability/integrity through MEK1/ERK1/2 because we reported that ERK1/2 activity is necessary for dynamic instability of interphase microtubules in RAS-transformed fibroblasts [34], and others have shown that microtubule stability results in multi-pole spindles [35], and breast cancer gene 1 (BRCA1)-ERK1/2 form complexes during mitosis [36, 37]
Our data suggest that RHAMM proteins control the structure of interphase and mitotic spindle microtubules and that these effects are driven by MEK1/ERK1/2 kinase activity
Summary
The interphase microtubule network is composed of ␣and -tubulin heterodimers that form tubules, which are highly dynamic structures participating in cell morphology/ polarity, signaling, migration, proliferation, and protein trafficking [1,2,3]. Expression of mutant RHAMM⌬163 defective in ERK1 binding in mouse embryonic fibroblasts, and blocking MEK1 activity collectively confirm that the effect of RHAMM⌬163 on interphase and mitotic spindle microtubules is mediated by ERK1/2 activity.
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