A Screen for PKN3 Substrates Reveals an Activating Phosphorylation of ARHGAP18.

Michal Dibus,Daniel Rösel,Jan Brábek

doi:10.3390/ijms21207769

Michal Dibus, Daniel Rösel + Show 1 more

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https://doi.org/10.3390/ijms21207769

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Abstract

Protein kinase N3 (PKN3) is a serine/threonine kinase implicated in tumor progression of multiple cancer types, however, its substrates and effector proteins still remain largely understudied. In the present work we aimed to identify novel PKN3 substrates in a phosphoproteomic screen using analog sensitive PKN3. Among the identified putative substrates we selected ARHGAP18, a protein from RhoGAP family, for validation of the screen and further study. We confirmed that PKN3 can phosphorylate ARHGAP18 in vitro and we also characterized the interaction of the two proteins, which is mediated via the N-terminal part of ARHGAP18. We present strong evidence that PKN3-ARHGAP18 interaction is increased upon ARHGAP18 phosphorylation and that the phosphorylation of ARHGAP18 by PKN3 enhances its GAP domain activity and contributes to negative regulation of active RhoA. Taken together, we identified new set of potential PKN3 substrates and revealed a new negative feedback regulatory mechanism of Rho signaling mediated by PKN3-induced ARHGAP18 activation.

Highlights

Protein kinase N3 (PKN3) is a serine/threonine kinase belonging to the PKN family of kinases that act downstream of small Rho GTPases
We found that PKN3 interacts with ARHGAP18 and we present strong evidence that PKN3 phosphorylation of ARHGAP18 leads to the activation of its GAP domain, resulting in a decrease of RhoA activity
The phosphoproteomic screen was performed in the lysate of MDA-MB-231 breast cancer cells expressing either PKN3 AS or PKN3 KD as a control

Summary

Introduction

PKN3 (protein kinase N3) is a serine/threonine kinase belonging to the PKN family of kinases that act downstream of small Rho GTPases. Downregulation of PKN3 was shown to block cell migration and formation of tubular structures in both 2D and 3D as a result of impaired actin reorganization [4,10]. Selective targeting of PKN3 expression in endothelial cells by systemic administration of small liposomal siRNA, Atu, impaired the formation of micro- and macro-metastases in lungs in both experimental and spontaneous metastasis mouse models [11]. PKN3 was shown to act downstream of activated PI3K (phosphatidylinositol 3-kinase) promoting the malignant progression of prostate cancer [8]. Downregulation of PKN3 expression led to impaired primary tumor growth and inhibition of metastasis in breast and prostate cancer [9]. The effect of PKN3 expression on regulation of cancer development was further highlighted in PKN3 knock-out mice that showed slower rates of leukemia development induced by the loss of PTEN (phosphatase and tensin homolog) [14] and a decrease in the number of secondary tumor sites [15]

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