Phosphoinositides (PIPs) are low-abundant yet essential phospholipids localized in the cytosolic leaflets of cellular membranes. A key function of PIPs is to serve as membrane identity markers, where they engage numerous types of PIP effector domains. Pleckstrin homology (PH) domain-containing proteins are the largest family of such “readers” of the PIP-based code of membrane identity, wherein PIP binding to PH domains controls subcellular location and protein activity toward downstream signaling events. PH domains often appear in multidomain proteins, linked to enzymatic or protein-protein interaction domains that can propagate signal transduction. Yet, there is a limited understanding of how all but a handful of the >250 PH domain-containing proteins transduce membrane recognition to downstream signaling.This dissertation describes the biomolecular characterization and protein engineering of an understudied, microtubule- and plasma membrane-localized, PH domain-containing protein, PLEKHA5 (Pleckstrin homology domain-containing family A, member 5). At the microtubule network, PLEKHA5 functions as an adaptor of an E3 ubiquitin ligase, the Anaphse Promoting Complex/Cyclosome (APC/C), to regulate the spatial organization of APC/C activity. Microtubule-localized proximity biotinylation tools reveal that PLEKHA5 promotes APC/C’s subcellular localization to microtubules and facilitates its activation, thus ensuring the timely turnover of key mitotic APC/C substrates and proper progression through mitosis. Although PLEKHA5 predominately localizes to the microtubule cytoskeleton in interphase, a portion of the protein translocate to the plasma membrane (PM) via interaction with PI(4,5)P2 specifically during mitosis, and the membrane association of PLEKHA5 is regulated by the phosphorylation status of a single serine near its PH domain. Harnessing this dynamic PM binding capability of PLEKHA5, we utilize protein engineering to develop a reversible, mitosis-specific protein recruitment platform at the PM we termed MARS (Mitosis-enabled Anchor-away/Recruiter System). We successfully apply MARS to recruit functional enzymes to generate signaling molecules at the PM in mitosis. In summary, the work presented in this dissertation highlights a link between the spatial organization of APC/C and the cellular functions of PLEKHA5 as a microtubule adaptor protein and develops a perturbation-free platform for conditional protein translocation capitalizing on the mitosis-specific plasma membrane localization of PLEKHA5.