# Phosphoinositide-Binding Proteins as Regulators of Ubiquitination and Wnt Signaling

> **NIH NIH R01** · CORNELL UNIVERSITY · 2022 · $307,716

## Abstract

Project Summary/Abstract
Phosphoinositides (PIPs) and ubiquitination are major systems that modulate signal transduction in space and
time. While their primary regulatory mechanisms are well characterized, secondary layers of regulation,
particularly those regulating crosstalk, have received less attention. We have identified a novel link between PIPs
and ubiquitination mediated by PLEKHA4, a pleckstrin homology (PH) domain-containing protein. We discovered
that this multi-domain protein forms large assemblies at PI(4,5)P2-rich regions of the plasma membrane, via a
unique combination of lipid- and protein-binding domains, and recruits the E3 ubiquitin ligase CUL3KLHL12 to such
structures. Surprisingly, this relocalization of CUL3KLHL12 is accompanied by a decrease in E3 ligase activity
toward a major substrate, Dishevelled-3 (DVL3), leading to DVL3 accumulation and increases in Wnt signaling,
in which DVL3 is a key intermediate. It remains unknown how, mechanistically, PLEKHA4 modulates CUL3KLHL12
activity, at both the molecular and functional levels. In this proposal we will test a novel sequestration model to
explain and understand these results. Our long-term research goal is to understand how PIP-sensing proteins
link membrane lipid composition to regulate signaling proteins in diverse physiological contexts. The objective of
this proposal is to understand mechanisms of how PLEKHA4 and its paralogs PLEKHA5/6/7 affect CUL3KLHL12
E3 ligase activity toward DVL3 and Wnt signaling. The central hypothesis guiding this work is that oligomeric
clusters of PLEKHA4/5/6/7 mediate sequestration of CUL3KLHL12 at the plasma membrane in an inactive state
and, via preventing DVL3 ubiquitination, act as positive regulators of Wnt signaling. We propose the following
aims to achieve our goals: (1) Elucidate molecular mechanisms governing PLEKHA4 regulation of
CUL3KLHL12-mediated DVL3 ubiquitination. We will test the sequestration model for PLEKHA4 function by
performing rescue of RNAi-induced phenotypes with PLEKHA4 constructs deficient in different molecular
functions, including membrane binding and oligomerization. We will also explore contributions of changes in
PI(4,5)P2 metabolism to PLEKHA4 function. (2) Determine the mechanistic basis for PLEKHA4’s
physiological effects on Wnt signaling in the Drosophila model. We found that a knockout of the single
ancestral fly PLEKHA4/5/6/7 homolog exhibits defects in Wnt/Wingless signaling. We will perform in vivo
structure–function studies to dissect the mechanisms contributing to these phenotypes. (3) Elucidate
specialization and conservation of function at the molecular and cellular levels within the PLEKHA4/5/6/7
family. We will test the hypothesis that the PLEKHA4/5/6/7 proteins can form multiple functional PLEKHA
complexes via hetero-oligomerization and differential degrees of membrane and protein affinities. In sum,
understanding how the PLEKHA4/5/6/7 proteins can read the dynamically changing PIP content of m...

## Key facts

- **NIH application ID:** 10458495
- **Project number:** 5R01GM131101-05
- **Recipient organization:** CORNELL UNIVERSITY
- **Principal Investigator:** Jeremy Baskin
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $307,716
- **Award type:** 5
- **Project period:** 2018-09-20 → 2023-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10458495

## Citation

> US National Institutes of Health, RePORTER application 10458495, Phosphoinositide-Binding Proteins as Regulators of Ubiquitination and Wnt Signaling (5R01GM131101-05). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10458495. Licensed CC0.

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