Alison J. Lin

Site-specific protein labeling by Sfp phosphopantetheinyl transferase

Sfp phosphopantetheinyl transferase covalently attaches small-molecule probes including biotin and various organic fluorophores to a specific serine residue in the peptidyl carrier protein (PCP) or a short 11-residue peptide tag ybbR through a phosphopantetheinyl linker. We describe here a protocol for site-specific protein labeling by Sfp-catalyzed protein post-translational modification that includes (i) expression and purification of Sfp, (ii) synthesis of small-molecule probe–CoA conjugates, (iii) construction of target protein fusions with PCP or the ybbR tag, (iv) labeling PCP- or ybbR-tagged target protein fusions in cell lysates and on live cell surfaces and (v) imaging fluorophore-labeled cell surface receptors by fluorescence microscopy. To follow this protocol, we advise that you allow 3 d for the expression and purification of Sfp phosphopantetheinyl transferase, 1 d for the synthesis and purification of the small-molecule probe–CoA conjugates as the substrates of Sfp, 3 d for the cloning of target protein genes as fusions to the PCP or the ybbR tag in the appropriate plasmids and another 3 d for transfecting cell lines with the plasmids and the expression of PCP- or ybbR-tagged proteins. Labeling of the PCP- or the ybbR-tagged proteins in cell lysates or on cell surfaces should require only 15–30 min.

Viral Mimicry of Cdc2/cyclin-dependent Kinase 1 Mediates Disruption of Nuclear Lamina during Human Cytomegalovirus Nuclear Egress

The nuclear lamina is a major obstacle encountered by herpesvirus nucleocapsids in their passage from the nucleus to the cytoplasm (nuclear egress). We found that the human cytomegalovirus (HCMV)-encoded protein kinase UL97, which is required for efficient nuclear egress, phosphorylates the nuclear lamina component lamin A/C in vitro on sites targeted by Cdc2/cyclin-dependent kinase 1, the enzyme that is responsible for breaking down the nuclear lamina during mitosis. Quantitative mass spectrometry analyses, comparing lamin A/C isolated from cells infected with viruses either expressing or lacking UL97 activity, revealed UL97-dependent phosphorylation of lamin A/C on the serine at residue 22 (Ser22). Transient treatment of HCMV-infected cells with maribavir, an inhibitor of UL97 kinase activity, reduced lamin A/C phosphorylation by approximately 50%, consistent with UL97 directly phosphorylating lamin A/C during HCMV replication. Phosphorylation of lamin A/C during viral replication was accompanied by changes in the shape of the nucleus, as well as thinning, invaginations, and discrete breaks in the nuclear lamina, all of which required UL97 activity. As Ser22 is a phosphorylation site of particularly strong relevance for lamin A/C disassembly, our data support a model wherein viral mimicry of a mitotic host cell kinase activity promotes nuclear egress while accommodating viral arrest of the cell cycle.

Subcellular Targeting and Agonist-induced Site-specific Phosphorylation of Endothelial Nitric-oxide Synthase

The endothelial isoform of nitric-oxide synthase (eNOS) undergoes a complex pattern of covalent modifications, including acylation with the fatty acids myristate and palmitate as well as phosphorylation on multiple sites. eNOS acylation is a key determinant for the reversible subcellular targeting of the enzyme to plasmalemmal caveolae. We transfected a series of hemagglutinin epitope-tagged eNOS mutant cDNAs deficient in palmitoylation (palm−) and/or myristoylation (myr−) into bovine aortic endothelial cells; after treatment with the eNOS agonists sphingosine 1-phosphate or vascular endothelial growth factor, the recombinant eNOS was immunoprecipitated using an antibody directed against the epitope tag, and patterns of eNOS phosphorylation were analyzed in immunoblots probed with phosphorylation state-specific eNOS antibodies. The wild-type eNOS underwent agonist-induced phosphorylation at serine 1179 (a putative site for phosphorylation by kinase Akt), but phosphorylation of the myr− eNOS at this residue was nearly abrogated; the palm− eNOS exhibited an intermediate phenotype. The addition of the CD8 transmembrane domain to the amino terminus of eNOS acylation-deficient mutants rescued the wild-type phenotype of robust agonist-induced serine 1179 phosphorylation. Thus, membrane targeting, but not necessarily acylation, is the critical determinant for agonist-promoted eNOS phosphorylation at serine 1179. In striking contrast to serine 1179, phosphorylation of eNOS at serine 116 was enhanced in the myr− eNOS mutant and was markedly attenuated in the CD8-eNOS membrane-targeted fusion protein. We conclude that eNOS targeting differentially affects eNOS phosphorylation at distinct sites in the protein and suggest that the inter-relationships of eNOS acylation and phosphorylation may modulate eNOS localization and activity and thereby influence NO signaling pathways in the vessel wall.

Atrial Natriuretic Peptide-initiated cGMP Pathways Regulate Vasodilator-stimulated Phosphoprotein Phosphorylation and Angiogenesis in Vascular Endothelium

Nitric oxide (NO)- and atrial natriuretic peptide (ANP)-initiated cGMP signaling cascades are important in the maintenance of cardiovascular homeostasis. The molecular signaling mechanisms downstream of cGMP are not well understood, however. We have used small interfering RNA (siRNA) approaches to specifically knock down a series of signaling proteins in bovine aortic endothelial cells, and we have combined biochemical analyses with physiological assays to investigate cGMP-mediated signal transduction pathways. Activation of particulate guanylate cyclase (GC-A) by ANP leads to a substantial, dose-dependent, rapid, and sustained increase in intracellular cGMP. In contrast, stimulation of soluble guanylate cyclase by NO yields only a weak and transient increase in cGMP. ANP-induced cGMP production is selectively suppressed by siRNA-mediated knockdown of GC-A. ANP greatly enhances the phosphorylation at Ser-239 of the vasodilator-stimulated phosphoprotein (VASP), a major substrate of cGMP-dependent protein kinase (PKG) that significantly influences actin dynamics. Moreover, the ANP-induced phosphorylation of VASP at Ser-239 is accompanied by increased actin stress fiber formation and enhanced endothelial tube formation. siRNA-mediated knockdown of GC-A, VASP, or PKG abolishes ANP-induced VASP Ser-239 phosphorylation, stress fiber formation, and endothelial tube formation. We have demonstrated similar findings in human umbilical vein endothelial cells, where ANP substantially enhances intracellular cGMP content, phosphorylation of VASP at Ser-239, and endothelial tube formation. Taken together, our findings suggest that ANP-mediated cGMP signal transduction pathways regulate PKG phosphorylation of VASP Ser-239 in endothelial cells, resulting in reorganization of the actin cytoskeleton and enhancement of angiogenesis.

Inactivation of Retinoblastoma Protein Does Not Overcome the Requirement for Human Cytomegalovirus UL97 in Lamina Disruption and Nuclear Egress

ABSTRACT Human cytomegalovirus (HCMV) encodes one conventional protein kinase, UL97. During infection, UL97 phosphorylates the retinoblastoma tumor suppressor protein (pRb) on sites ordinarily phosphorylated by cyclin-dependent kinases (CDK), inactivating the ability of pRb to repress host genes required for cell cycle progression to S phase. UL97 is important for viral DNA synthesis in quiescent cells, but this function can be replaced by human papillomavirus type 16 E7, which targets pRb for degradation. However, viruses in which E7 replaces UL97 are still defective for virus production. UL97 is also required for efficient nuclear egress of viral nucleocapsids, which is associated with disruption of the nuclear lamina during infection, and phosphorylation of lamin A/C on serine 22, which antagonizes lamin polymerization. We investigated whether inactivation of pRb might overcome the requirement of UL97 for these roles, as pRb inactivation induces CDK1, and CDK1 phosphorylates lamin A/C on serine 22. We found that lamin A/C serine 22 phosphorylation during HCMV infection correlated with expression of UL97 and was considerably delayed in UL97-null mutants, even when E7 was expressed. E7 failed to restore gaps in the nuclear lamina seen in wild-type but not UL97-null virus infections. In electron microscopy analyses, a UL97-null virus expressing E7 was as impaired as a UL97-null mutant in cytoplasmic accumulation of viral nucleocapsids. Our results demonstrate that pRb inactivation is insufficient to restore efficient viral nuclear egress of HCMV in the absence of UL97 and instead argue further for a direct role of UL97 in this stage of the infectious cycle.

MARCKS protein mediates hydrogen peroxide regulation of endothelial permeability

Impairment of endothelial barrier function is implicated in many vascular and inflammatory disorders. One prevalent mechanism of endothelial dysfunction is an increase in reactive oxygen species under oxidative stress. Previous reports have demonstrated that hydrogen peroxide (H2O2), a highly stable reactive oxygen species that modulates physiological signaling pathways, also enhances endothelial permeability, but the mechanism of this effect is unknown. Here, we identify the actin-binding protein myristoylated alanine-rich C-kinase substrate (MARCKS) as a key mediator of the H2O2-induced permeability change in bovine aortic endothelial cells. MARCKS knockdown and H2O2 treatment alter the architecture of the actin cytoskeleton in endothelial cells, and H2O2 induces the phosphorylation and translocation of MARCKS from the cell membrane to the cytosol. Using pharmacological inhibitors and small interference RNA constructs directed against specific proteins, we uncover a signaling cascade from Rac1 to Abl1, phospholipase Cγ1, and PKCδ that is triggered by H2O2 and leads to MARCKS phosphorylation. Our findings establish a distinct role for MARCKS in the regulation of H2O2-induced permeability change in endothelial cells, and suggest potential new therapeutic targets for the treatment of disorders involving oxidative stress and altered endothelial permeability.