CGMP-dependent protein kinase I gamma encodes a nuclear localization signal that regulates nuclear compartmentation and function

Abstract

cGMP-dependent protein kinase I (PKGI) plays an important role in regulating how cGMP specifies vascular smooth muscle cell (SMC) phenotype. Although studies indicate that PKGI nuclear localization controls how cGMP regulates gene expression in SMC, information about the mechanisms that regulate PKGI nuclear compartmentation and its role in directly regulating cell phenotype is limited. Here we characterize a nuclear localization signal sequence (NLS) in PKGIγ, a proteolytically cleaved PKGI kinase fragment that translocates to the nucleus of SMC. Immuno-localization studies using cells expressing native and NLS-mutant PKGIγ, and treated with a small molecule nuclear transport inhibitor, indicated that PKGIγ encodes a constitutively active NLS that requires importin α and β for regulation of its compartmentation. Moreover, studies utilizing a genetically encoded nuclear phospho-CREB biosensor probe and fluorescence lifetime imaging microscopy demonstrated that this NLS controls PKGIγ nuclear function. In addition, although cytosolic PKGIγ-activity was observed to stimulate MAPK/ERK-mediated nuclear CREB signaling in SMC, NLS-mediated PKGIγ nuclear activity alone was determined to increase the expression of differentiation marker proteins in these cells. These results indicate that NLS-mediated nuclear PKGIγ localization plays an important role in how PKGI regulates vascular SMC phenotype.