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

> **NIH NIH R01** · CORNELL UNIVERSITY · 2022 · $10,660

## Abstract

PROJECT SUMMARY
Parent Grant R01 GM131101
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...

## Key facts

- **NIH application ID:** 10589550
- **Project number:** 3R01GM131101-04S1
- **Recipient organization:** CORNELL UNIVERSITY
- **Principal Investigator:** Jeremy Baskin
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $10,660
- **Award type:** 3
- **Project period:** 2018-09-20 → 2023-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10589550

## Citation

> US National Institutes of Health, RePORTER application 10589550, Phosphoinositide-Binding Proteins as Regulators of Ubiquitination and Wnt Signaling (3R01GM131101-04S1). Retrieved via AI Analytics 2026-05-27 from https://api.ai-analytics.org/grant/nih/10589550. Licensed CC0.

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