The PP2A-B56 Binding Site LxxIxE Contributes to Asp-Mediated Spindle Pole Stability.

Abstract

The organization of microtubules into a mitotic spindle is critical for animal cell proliferation and involves the cooperation of hundreds of proteins whose molecular roles and regulation are not fully understood. The protein product of the Drosophila gene abnormal spindle, Asp, is a microtubule-associated protein required for correct mitotic spindle formation. To better understand the contribution of Asp to microtubule organization during spindle formation, we reverse-engineered flies to express a version of Asp (Asp^LIE), predicted to have lost its ability to bind the phosphatase trimer PP2A-B56. We demonstrated that the Asp^LIE mutation reduced an interaction with the Drosophila PP2A-B56 regulatory subunit Widerborst (Wdb), as well as other proteins with known roles in spindle formation. Asp^LIE flies exhibited less robust microtubule minus-end cohesion at neural stem cell spindle poles, which was accompanied by a substantial developmental delay but no microcephaly. Predictive structural modeling suggests that the presence of Wdb alters the conformation of an Asp interaction with a tubulin dimer in a manner similar to that of the Asp^LIE mutation. Protein localization in the Drosophila embryo, in addition to in vitro microtubule organization experiments, suggests that a role of PP2A may be to prevent Asp from contributing to microtubule cross-linking at spindle microtubule plus ends. Together, these findings add new insights to mechanisms underlying microtubule organization within the mitotic spindle.