# Regulation of Innate Immunity by the Hippo Kinases

> **NIH NIH R21** · WAYNE STATE UNIVERSITY · 2024 · $229,955

## Abstract

Project Summary
The Hippo pathway is conserved from yeasts to humans. In mammals, this signaling pathway controls tissue
development by balancing cell proliferation and death. The Hippo kinases, MST1 and MST2, transduce signals
via phosphorylation of the scaffold protein MOB1 and the downstream kinase LAST1. Animal models show that
tissue-specific Mst1/2 gene knockouts lead to abnormal organ development and tumor formation. In addition to
its well-known functions in development and cancer, recent studies found the Hippo pathway is targeted and
manipulated by bacterial pathogens, suggesting a role of this conserved host pathway in bacterial pathogenesis.
Notably, individuals with loss-of-function mutations in the MST1 gene are immunocompromised. Consistent with
this observation in humans, our lab knocked out both Mst1 and Mst2 genes in macrophage and found that these
macrophages are highly susceptible to infection by the human pathogen Legionella pneumophila, highlighting
an emerging role of the Hippo kinases in immunity against infection. Using the Mst1/2 double knockout cell model,
we further discovered that MST1/2 are the key regulators for immune gene expression and programmed cell
death during infection. Importantly, upon challenge of virulent L. pneumophila, MST1/2 full-length proteins are
proteolytically cleaved, resulting in MST1/2 fragments (MST1/2-NT) that contain the kinase domains but are
separated from the regulatory domains. In biochemical assays, MST1/2-NT, surprisingly, no longer
phosphorylate their cognate substrate, MOB1, but exhibit altered substrate specificity for protein phosphorylation.
In cell-based assays, MST1/2-NT production contributes to activation of apoptotic cell death that is critical for
restricting virulent L. pneumophila. Based on the preliminary data collected in our lab, we propose a model that,
upon infection, macrophages trigger cleavage of MST1/2 to activate an inflammatory Hippo signaling mediated
by the proapoptotic MST1/2-NT as a defense mechanism. The objective of the proposed study is to dissect the
importance of this novel inflammatory Hippo signaling. In Aim 1, we will use an innovative technique,
Thiophosphorylation Proteome Screen (TPS), that allows us to screen more than 21,000 human proteins for
direct substrates of full-length MST1/2 and MST1/2-NT. We will use the established substrate validation methods
which include in vitro and cell-based assays to validate genuine substrates of these kinases. This aim will yield
a comprehensive phosphoproteome controlled by the Hippo kinases that is fundamental for understanding both
the canonical and inflammatory Hippo signaling. In Aim 2, we will focus on how MST1/2 influence gene
expression and communication in immune cells. Specifically, we discovered that expression of the complement
protein C1q is depleted in Mst1/2 double knockout macrophages. We will use genetic and cell-based assays to
elucidate the molecular mechanism by which MST1/2 regulate C1q, ...

## Key facts

- **NIH application ID:** 10867703
- **Project number:** 1R21AI182688-01
- **Recipient organization:** WAYNE STATE UNIVERSITY
- **Principal Investigator:** Pei-Chung Lee
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $229,955
- **Award type:** 1
- **Project period:** 2024-05-22 → 2026-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10867703

## Citation

> US National Institutes of Health, RePORTER application 10867703, Regulation of Innate Immunity by the Hippo Kinases (1R21AI182688-01). Retrieved via AI Analytics 2026-05-28 from https://api.ai-analytics.org/grant/nih/10867703. Licensed CC0.

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