Formin homology domain protein (FHOD1) is a cyclic GMP-dependent protein kinase I-binding protein and substrate in vascular smooth muscle cells.

Howard K. Surks,Yuepeng Wang,Michael E. Mendelsohn,Mohamad R. El-Zaru

doi:10.1074/jbc.m313823200

Howard K. Surks, Yuepeng Wang + Show 2 more

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https://doi.org/10.1074/jbc.m313823200

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Abstract

Cyclic GMP-dependent protein kinase I (PKGI) mediates vascular relaxation by nitric oxide and related nitrovasodilators and inhibits vascular smooth muscle cell (VSMC) migration. To identify VSMC proteins that interact with PKGI, the N-terminal protein interaction domain of PKGIalpha was used to screen a yeast two-hybrid human aortic cDNA library. The formin homology (FH) domain-containing protein, FHOD1, was found to interact with PKGIalpha in this screen. FH domain-containing proteins bind Rho-family GTPases and regulate actin cytoskeletal dynamics, cell migration, and gene expression. Antisera to FHOD1 were raised and used to characterize FHOD1 expression and distribution in vascular cells. FHOD1 is highly expressed in human coronary artery, aortic smooth muscle cells, and in human arterial and venous endothelial cells. In glutathione S-transferase pull-down experiments, the FHOD1 C terminus (amino acids 964-1165) binds full-length PKGI. Both in vitro and intact cell studies demonstrate that the interaction between FHOD1 and PKGI is decreased 3- to 5-fold in the presence of the PKG activator, 8Br-cGMP. Immunofluorescence studies of human VSMC show that FHOD1 is cytoplasmic and is concentrated in the perinuclear region. PKGI also directly phosphorylates FHOD1, and studies with wild-type and mutant FHOD1-derived peptides identify Ser-1131 in the FHOD1 C terminus as the unique PKGI phosphorylation site in FHOD1. These studies demonstrate that FHOD1 is a PKGI-interacting protein and substrate in VSMCs and show that cyclic GMP negatively regulates the FHOD1-PKGI interaction. Based on the known functions of FHOD1, the data are consistent with a role for FHOD1 in cyclic GMP-dependent inhibition of VSMC stress fiber formation and/or migration.

Highlights

A number of laboratories have sought proteins capable of interacting with PKGI isoforms (PKGI-interacting proteins, cGMP-dependent protein kinase-anchoring proteins, cGMP-dependent protein kinase-interacting proteins (GKIPs))
Consistent with this notion, we recently identified the regulator of G-protein signaling (RGS2) as a second GKIP that is important in regusaline; FHOD1, formin-homology-2 domain-containing protein; PP1M, myosin phosphatase
FHOD1 was found principally in the Triton-soluble vascular smooth muscle cell fractions, and this distribution was not altered by exposure of cells to either angiotensin II or 8Br-cGMP (Fig. 2B)

Summary

Introduction

A number of laboratories have sought proteins capable of interacting with PKGI isoforms (PKGI-interacting proteins, cGMP-dependent protein kinase-anchoring proteins, cGMP-dependent protein kinase-interacting proteins (GKIPs)). The LZ of PKGI␣, but not the homologous LZ in the amino termini of PKGI␤ or PKGII, binds PP1M [13], suggesting additional molecular determinants of specificity are contained in these domains. Recent studies show that the N-terminal LZ mediates the interaction of PKGI␤ with the IP3Rassociated cGMP kinase substrate (IRAG) protein [17], suggesting that the N-terminal LZs of the PKGI isoforms may contribute more generally to protein targeting. Consistent with this notion, we recently identified the regulator of G-protein signaling (RGS2) as a second GKIP that is important in regusaline; FHOD1, formin-homology-2 domain-containing protein; PP1M, myosin phosphatase. We report the identification of a new GKIP and PKGI substrate, the formin homology (FH) domain-containing protein FHOD1 [19, 20]

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