The Wnt pathway protein Dvl1 targets somatostatin receptor 2 for lysosome-dependent degradation.

The Somatostatin receptor 2 (Sstr2) is a heterotrimeric G protein-coupled receptor that is highly expressed in neuroendocrine tumors and is a common pharmacological target for intervention. Unfortunately, not all neuroendocrine tumors express Sstr2, and Sstr2 expression can be downregulated with prolonged agonist use. Sstr2 is rapidly internalized following agonist stimulation and, in the short term, is quantitatively recycled back to the plasma membrane. However, mechanisms controlling steady state expression of Sstr2 in the absence of agonist are less well described. Here, we show that Sstr2 interacts with the Wnt pathway protein Dvl1 in a ligand-independent manner to target Sstr2 for lysosomal degradation. Interaction of Sstr2 with Dvl1 does not affect receptor internalization, recycling, or signaling to adenylyl cyclase but does suppress agonist-stimulated ERK1/2 activation. Importantly, Dvl1-dependent degradation of Sstr2 can be stimulated by overexpression of Wnts and treatment of cells with Wnt pathway inhibitors can boost Sstr2 expression in neuroendocrine tumor cells. Taken together, this study identifies for the first time a mechanism that targets Sstr2 for lysosomal degradation that is independent of Sstr2 agonist and can be potentiated by Wnt ligand. Intervention in this signaling mechanism has the potential to elevate Sstr2 expression in neuroendocrine tumors and enhance Sstr2-directed therapies.

Src stimulates Abl-dependent phosphorylation of the guanine exchange factor Net1A to promote its cytosolic localization and cell motility.

The neuroepithelial cell transforming gene 1 (Net1) is a guanine nucleotide exchange factor for the small GTPase RhoA that promotes cancer cell motility and metastasis. Two isoforms of Net1 exist, Net1 and Net1A, both of which are sequestered in the nucleus in quiescent cells to prevent aberrant RhoA activation. Many cell motility stimuli drive cytosolic relocalization of Net1A, but mechanisms controlling this event are not fully understood. Here, we demonstrate that epithelial growth factor stimulates protein kinase Src- and Abl1-dependent phosphorylation of Net1A to promote its cytosolic localization. We show that Abl1 efficiently phosphorylates Net1A on Y373, and that phenylalanine substitution of Y373 prevents Net1A cytosolic localization. Furthermore, we found that Abl1-driven cytosolic localization of Net1A does not require S52, which is a phosphorylation site of a different kinase, c-Jun N-terminal kinase, that inhibits nuclear import of Net1A. However, we did find that MKK7-stimulated cytosolic localization of Net1A does require Y373. We also demonstrate that aspartate substitution at Y373 is sufficient to promote Net1A cytosolic accumulation, and expression of Net1A Y373D potentiates epithelial growth factor-stimulated RhoA activation, downstream myosin light chain 2 phosphorylation, and F-actin accumulation. Moreover, we show that expression of Net1A Y373D in breast cancer cells also significantly increases cell motility and Matrigel invasion. Finally, we show that Net1A is required for Abl1-stimulated cell motility, which is rescued by expression of Net1A Y373D, but not Net1A Y373F. Taken together, this work demonstrates a novel mechanism controlling Net1A subcellular localization to regulate RhoA-dependent cell motility and invasion.

Acetylation of the RhoA GEF Net1A controls its subcellular localization and activity.

Net1 isoform A (Net1A) is a RhoA GEF that is required for cell motility and invasion in multiple cancers. Nuclear localization of Net1A negatively regulates its activity, and we have recently shown that Rac1 stimulates Net1A relocalization to the plasma membrane to promote RhoA activation and cytoskeletal reorganization. However, mechanisms controlling the subcellular localization of Net1A are not well understood. Here, we show that Net1A contains two nuclear localization signal (NLS) sequences within its N-terminus and that residues surrounding the second NLS sequence are acetylated. Treatment of cells with deacetylase inhibitors or expression of active Rac1 promotes Net1A acetylation. Deacetylase inhibition is sufficient for Net1A relocalization outside the nucleus, and replacement of the N-terminal acetylation sites with arginine residues prevents cytoplasmic accumulation of Net1A caused by deacetylase inhibition or EGF stimulation. By contrast, replacement of these sites with glutamine residues is sufficient for Net1A relocalization, RhoA activation and downstream signaling. Moreover, the N-terminal acetylation sites are required for rescue of F-actin accumulation and focal adhesion maturation in Net1 knockout MEFs. These data indicate that Net1A acetylation regulates its subcellular localization to impact on RhoA activity and actin cytoskeletal organization.

The PDZ Domain Protein SYNJ2BP Regulates GRK-Dependent Sst2A Phosphorylation and Downstream MAPK Signaling.

The somatostatin receptor 2A (SST2) is a G-protein-coupled receptor (GPCR) that is expressed in neuroendocrine tissues within the gastrointestinal tract and brain, and is commonly overexpressed in many neuroendocrine tumors. Moreover, SST2 agonists are used clinically as the primary pharmacological treatment to suppress excess hormone secretion in a variety of neuroendocrine tumors. Despite its wide clinical use, mechanisms controlling the trafficking and signaling of SST2 are not fully understood. SST2 contains a C-terminal post-synaptic density 95, Drosophila discs large, zona-occludens 1 (PDZ) domain-binding motif that has been shown to interact with 3 different PDZ domain-containing proteins. However, the consequences of these interactions are not well understood, nor is it known whether additional PDZ domain proteins interact with SST2. Through unbiased screening we have identified 10 additional PDZ domain proteins that interact with SST2. We chose one of these, SYNJ2BP, for further study. We observed that SYNJ2BP interacted with SST2 in an agonist-dependent manner, and that this required the PDZ binding site of SST2. Importantly, overexpression of SYNJ2BP enhanced ligand-stimulated receptor internalization. Mechanistically, SYNJ2BP interacted with G-protein-coupled receptor kinase 2 (GRK2) and promoted GRK-dependent phosphorylation of the receptor after somatostatin stimulation. Interaction with GRK2 required the C-terminus of SYNJ2BP. Binding to SYNJ2BP did not affect the ability of SST2 to suppress 3',5'-cyclic adenosine 5'-monophosphate production, but was required for optimal agonist-stimulated extracellularly regulated kinase 1/2 activation. These data indicated that SYNJ2BP is an SST2-interacting protein that modulates agonist-stimulated receptor regulation and downstream signaling.

Mutagenic analysis of Cox11 of Rhodobacter sphaeroides: insights into the assembly of Cu(B) of cytochrome c oxidase.

The Cu(I) chaperone Cox11 is required for the insertion of Cu(B) into cytochrome c oxidase (CcO) of mitochondria and many bacteria, including Rhodobacter sphaeroides. Exploration of the copper binding stoichiometry of R. sphaeroides Cox11 led to the finding that an apparent tetramer of both mitochondrial and bacterial Cox11 binds more copper than the sum of the dimers, providing another example of the flexibility of copper binding by Cu(I)-S clusters. Site-directed mutagenesis has been used to identify components of Cox11 that are not required for copper binding but are absolutely required for the assembly of Cu(B), including conserved Cys-35 and Lys-123. In contrast to earlier proposals, Cys-35 is not required for dimerization of Cox11 or for copper binding. These findings, and the location of Cys-35 at the C-terminus of the predicted transmembrane helix and thereby close to the surface of the membrane, allow a proposal that Cys-35 is involved in the transfer of copper from the Cu(I) cluster of Cox11 to the Cu(B) ligands His-333 and His-334 during the folding of CcO subunit I. Lys-123 is located near the Cu(I) cluster of Cox11, in an area otherwise devoid of charged residues. From the analysis of several Cox11 mutants, including K123E, -L, and -R, we conclude that a previous proposal that Lys-123 provides charge balance for the stabilization of the Cu(I) cluster is unlikely to account for its absolute requirement for Cox11 function. Rather, consideration of the properties of Lys-123 and the apparent specificity of Cox11 suggest that Lys-123 plays a role in the interaction of Cox11 with its target.

Regulation of Somatostatin Receptor 2 Trafficking by C-Tail Motifs and the Retromer.

The Gi-coupled somatostatin receptor 2 (SST2) is a G protein-coupled receptor (GPCR) that mediates many of somatostatin's neuroendocrine actions. Upon stimulation, SST2 is rapidly internalized and transported to early endosomes before being recycled to the plasma membrane. However, little is known about the intracellular itinerary of SST2 after it moves to the early endosomal compartment or the cytoplasmic proteins that regulate its trafficking. As postsynaptic density protein/discs large 1/zonula occludens-1 (PDZ) domain interactions often regulate the trafficking and signaling potential of GPCRs, we examined the role of the SST2 PDZ ligand and additional C-terminal residues in controlling its intracellular trafficking. We determined that SST2 can recycle to the plasma membrane via multiple pathways, including a LAMP1/Rab7-positive late endosome to the trans-Golgi network (TGN) pathway. Trafficking from the late endosome to the TGN is often regulated by the retromer complex of endosomal coat proteins, and disrupting the retromer components sorting nexins 1/2 inhibits the budding of SST2 from late endosomes. Moreover, trafficking through the late endosomal/TGN pathway is dependent on an intact PDZ ligand and C-terminal tail, as truncating either the 3 or 10 C-terminal amino acids of SST2 alters the pathway through which it recycles to the plasma membrane. Moreover, addition of these amino acids to a heterologous receptor is sufficient to redirect it from a degradation pathway to a recycling itinerary. Our results demonstrate that endosomal trafficking of SST2 is dependent on numerous regulatory mechanisms controlled by its C terminus and the retromer machinery.

p21 activated kinase 5 activates Raf-1 and targets it to mitochondria.

Raf-1 is an important effector of Ras mediated signaling and is a critical regulator of the ERK/MAPK pathway. Raf-1 activation is controlled in part by phosphorylation on multiple residues, including an obligate phosphorylation site at serine 338. Previously PAK1 and casein kinase II have been implicated as serine 338 kinases. To identify novel kinases that phosphorylate this site, we tested the ability of group II PAKs (PAKs 4-6) to control serine 338 phosphorylation. We observed that all group II PAKs were efficient serine 338 kinases, although only PAK1 and PAK5 significantly stimulated Raf-1 kinase activity. We also showed that PAK5 forms a tight complex with Raf-1 in the cell, but not A-Raf or B-Raf. Importantly, we also demonstrated that the association of Raf-1 with PAK5 targets a subpopulation of Raf-1 to mitochondria. These data indicate that PAK5 is a potent regulator of Raf-1 activity and may control Raf-1 dependent signaling at mitochondria.

Timing is everything: Rac1 controls Net1A localization to regulate cell adhesion.

Cell adhesion to the extracellular matrix elicits a temporal reorganization of the actin cytoskeleton that is regulated first by Rac1 and later by RhoA. The signaling mechanisms controlling late stage RhoA activation are incompletely understood. Net1A is a RhoA/RhoB-specific guanine nucleotide exchange factor that is required for cancer cell motility. The ability of Net1A to stimulate RhoA activation is negatively regulated by nuclear sequestration. However, mechanisms controlling the plasma membrane localization of Net1A had not previously been reported. Recently we have shown that Rac1 activation stimulates plasma membrane relocalization and activation of Net1A. Net1A relocalization is independent of its catalytic activity and does not require its C-terminal pleckstrin homology or PDZ interacting domains. Rac1 activation during cell adhesion stimulates a transient relocalization of Net1A that is terminated by proteasomal degradation of Net1A. Importantly, plasma membrane localization of Net1A is required for efficient myosin light chain phosphorylation, focal adhesion maturation, and cell spreading. These data show for the first time a physiological mechanism controlling Net1A relocalization from the nucleus. They also demonstrate a previously unrecognized role for Net1A in controlling actomyosin contractility and focal adhesion dynamics during cell adhesion.

Rho GTPase-independent regulation of mitotic progression by the RhoGEF Net1.

Neuroepithelial transforming gene 1 (Net1) is a RhoA-subfamily-specific guanine nucleotide exchange factor that is overexpressed in multiple human cancers and is required for proliferation. Molecular mechanisms underlying its role in cell proliferation are unknown. Here we show that overexpression or knockdown of Net1 causes mitotic defects. Net1 is required for chromosome congression during metaphase and generation of stable kinetochore microtubule attachments. Accordingly, inhibition of Net1 expression results in spindle assembly checkpoint activation. The ability of Net1 to control mitosis is independent of RhoA or RhoB activation, as knockdown of either GTPase does not phenocopy effects of Net1 knockdown on nuclear morphology, and effects of Net1 knockdown are effectively rescued by expression of catalytically inactive Net1. We also observe that Net1 expression is required for centrosomal activation of p21-activated kinase and its downstream kinase Aurora A, which are critical regulators of centrosome maturation and spindle assembly. These results identify Net1 as a novel regulator of mitosis and indicate that altered expression of Net1, as occurs in human cancers, may adversely affect genomic stability.

Regulation of focal adhesion kinase activation, breast cancer cell motility, and amoeboid invasion by the RhoA guanine nucleotide exchange factor Net1.

Net1 is a RhoA guanine nucleotide exchange factor (GEF) that is overexpressed in a subset of human cancers and contributes to cancer cell motility and invasion in vitro. However, the molecular mechanism accounting for its role in cell motility and invasion has not been described. In the present work, we show that expression of both Net1 isoforms in breast cancer cells is required for efficient cell motility. Although loss of Net1 isoform expression only partially blocks RhoA activation, it inhibits lysophosphatidic acid (LPA)-stimulated migration as efficiently as knockdown of RhoA itself. However, we demonstrate that the Net1A isoform predominantly controls myosin light-chain phosphorylation and is required for trailing edge retraction during migration. Net1A interacts with focal adhesion kinase (FAK), localizes to focal adhesions, and is necessary for FAK activation and focal adhesion maturation during cell spreading. Net1A expression is also required for efficient invasion through a Matrigel matrix. Analysis of invading cells demonstrates that Net1A is required for amoeboid invasion, and loss of Net1A expression causes cells to shift to a mesenchymal phenotype characterized by high ß1-integrin activity and membrane type 1 matrix metalloproteinase (MT1-MMP) expression. These results demonstrate a previously unrecognized role for the Net1A isoform in controlling FAK activation during planar cell movement and amoeboid motility during extracellular matrix (ECM) invasion.

Rac1 controls the subcellular localization of the Rho guanine nucleotide exchange factor Net1A to regulate focal adhesion formation and cell spreading.

RhoA is overexpressed in human cancer and contributes to aberrant cell motility and metastatic progression; however, regulatory mechanisms controlling RhoA activity in cancer are poorly understood. Neuroepithelial transforming gene 1 (Net1) is a RhoA guanine nucleotide exchange factor that is overexpressed in human cancer. It encodes two isoforms, Net1 and Net1A, which cycle between the nucleus and plasma membrane. Net1 proteins must leave the nucleus to activate RhoA, but mechanisms controlling the extranuclear localization of Net1 isoforms have not been described. Here, we show that Rac1 activation causes relocalization of Net1 isoforms outside the nucleus and stimulates Net1A catalytic activity. These effects do not require Net1A catalytic activity, its pleckstrin homology domain, or its regulatory C terminus. We also show that Rac1 activation protects Net1A from proteasome-mediated degradation. Replating cells on collagen stimulates endogenous Rac1 to relocalize Net1A, and inhibition of proteasome activity extends the duration and magnitude of Net1A relocalization. Importantly, we demonstrate that Net1A, but not Net1, is required for cell spreading on collagen, myosin light chain phosphorylation, and focal adhesion maturation. These data identify the first physiological mechanism controlling the extranuclear localization of Net1 isoforms. They also demonstrate a previously unrecognized role for Net1A in regulating cell adhesion.

Interaction of the RhoA exchange factor Net1 with discs large homolog 1 protects it from proteasome-mediated degradation and potentiates Net1 activity.

Net1 is a nuclear Rho guanine nucleotide exchange factor that is specific for the RhoA subfamily of small G proteins. Truncated forms of Net1 are transforming in NIH3T3 cells, and this activity requires cytoplasmic localization of Net1 as well as the presence of a COOH-terminal PDZ binding site. We have previously shown that Net1 interacts with PDZ domain-containing proteins within the Discs Large (Dlg) family and relocalizes them to the nucleus. In the present work, we demonstrate that Net1 binds directly to the first two PDZ domains of Dlg1 and that both PDZ domains are required for maximal interaction in cells. Furthermore, we show that Net1 is an unstable protein in MCF7 breast epithelial cells and that interaction with Dlg1 significantly enhances Net1 stability. Stabilization by Dlg1 significantly increases the ability of Net1 to stimulate RhoA activation in cells. The stability of endogenous Net1 is strongly enhanced by cell-cell contact, and this correlates with a dramatic increase in the interaction between Net1 and Dlg1. Importantly, disruption of E-cadherin-mediated cell contacts, either by depletion of external calcium or by treatment with transforming growth factor beta, leads to a rapid loss of the interaction between Net1 and Dlg1 and a subsequent increase in the ubiquitylation of Net1. These results indicate that Net1 requires interaction with PDZ domain proteins, such as Dlg1, to protect it from proteasome-mediated degradation and to maximally stimulate RhoA and that this interaction is regulated by cell-cell contact.

A bacterial cytotoxin identifies the RhoA exchange factor Net1 as a key effector in the response to DNA damage.

BACKGROUND: Exposure of adherent cells to DNA damaging agents, such as the bacterial cytolethal distending toxin (CDT) or ionizing radiations (IR), activates the small GTPase RhoA, which promotes the formation of actin stress fibers and delays cell death. The signalling intermediates that regulate RhoA activation and promote cell survival are unknown. PRINCIPAL FINDINGS: We demonstrate that the nuclear RhoA-specific Guanine nucleotide Exchange Factor (GEF) Net1 becomes dephosphorylated at a critical inhibitory site in cells exposed to CDT or IR. Expression of a dominant negative Net1 or Net1 knock down by iRNA prevented RhoA activation, inhibited the formation of stress fibers, and enhanced cell death, indicating that Net1 activation is required for this RhoA-mediated responses to genotoxic stress. The Net1 and RhoA-dependent signals involved activation of the Mitogen-Activated Protein Kinase p38 and its downstream target MAPK-activated protein kinase 2. SIGNIFICANCE: Our data highlight the importance of Net1 in controlling RhoA and p38 MAPK mediated cell survival in cells exposed to DNA damaging agents and illustrate a molecular pathway whereby chronic exposure to a bacterial toxin may promote genomic instability.

The nuclear RhoA exchange factor Net1 interacts with proteins of the Dlg family, affects their localization, and influences their tumor suppressor activity.

Net1 is a RhoA-specific guanine nucleotide exchange factor which localizes to the nucleus at steady state. A deletion in its N terminus redistributes the protein to the cytosol, where it activates RhoA and can promote transformation. Net1 contains a PDZ-binding motif at the C terminus which is essential for its transformation properties. Here, we found that Net1 interacts through its PDZ-binding motif with tumor suppressor proteins of the Dlg family, including Dlg1/SAP97, SAP102, and PSD95. The interaction between Net1 and its PDZ partners promotes the translocation of the PDZ proteins to nuclear subdomains associated with PML bodies. Interestingly, the oncogenic mutant of Net1 is unable to shuttle the PDZ proteins to the nucleus, although these proteins still associate as clusters in the cytosol. Our results suggest that the ability of oncogenic Net1 to transform cells may be in part related to its ability to sequester tumor suppressor proteins like Dlg1 in the cytosol, thereby interfering with their normal cellular function. In agreement with this, the transformation potential of oncogenic Net1 is reduced when it is coexpressed with Dlg1 or SAP102. Together, our results suggest that the interaction between Net1 and Dlg1 may contribute to the mechanism of Net1-mediated transformation.

Characterization of the biochemical and transforming properties of the neuroepithelial transforming protein 1.

Rho family small G proteins are key regulators of cytoskeletal organization and oncogenic transformation whose activation is controlled by a family of proteins known as guanine nucleotide exchange factors (GEFs). In this work we have characterized the structural and biological determinants for cytoskeletal regulation and cell transformation by the neuroepithelioma transforming gene 1 (NET1), which is a GEF specific for RhoA, but not Cdc42 or Rac1. Previously it was shown that the biological activity and nuclear localization of NET1 is controlled by its amino terminus. Here we demonstrate that the amino terminus of NET1 does not function as cis-acting autoinhibitory domain, nor does it affect the ability of full-length NET1 to stimulate actin stress fiber formation. We also show that the nuclear localization of NET1 is controlled by two separate domains within its amino terminus, only one of which contains the previously identified NLS sequences. Importantly, we find that the ability of NET1 to stimulate actin stress fiber formation does not correlate with its transforming activity, because NET1 proteins that potently stimulate stress fiber formation do not transform cells. Furthermore, the presence of a potential PDZ binding site in the C terminus of NET1 is critical to its ability to transform cells, but is not required for enzymatic activity or for effects on the actin cytoskeleton. Thus, these data highlight a divergence between the ability of NET1 to stimulate cytoskeletal reorganization and to transform cells, and implicate the interaction with PDZ domain-containing proteins as critical to NET1-dependent transformation.

Functional analysis of the domains in Cox11.

Cox11 is an intrinsic mitochondrial membrane protein essential for the assembly of an active cytochrome c oxidase complex. Cox11 is tethered to the mitochondrial inner membrane by a single transmembrane helix. Domain mapping was carried out to determine the functional segments of the Cox11 protein. The C-terminal 189 residue Cu(I)-binding domain is shown to be exposed within the mitochondrial intermembrane space. This orientation was demonstrated by the proteolytic susceptibility of a C-terminal Myc epitope tag in mitoplasts but not intact mitochondria. Fusion of the N terminus of Cox11 to the matrix ribosomal protein Rsm22 results in a functional protein capable of suppressing the respiratory defect of both Deltacox11 cells and Deltarsm22 cells. The functionality of the fusion protein suggests that the Cox11 N terminus projects into the matrix. The fusion of the C-terminal segment of Cox11 to Rsm22 resembles a naturally occurring fusion of Cox11 in Schizosaccharomyces pombe to a sequence homologous to the Saccharomyces cerevisiae Rsm22. Studies on a series of SCO1/COX11 chimeras reveal that the matrix domain of Cox11 lacks a specific function, whereas the Cu(I) binding/donating function requires the yeast Cox11 sequence. The Cu(I)-binding domain from human Cox11 cannot functionally replace the yeast sequence. The copper domain of Cox11 may be an important docking motif for Cox1 or a Cox1-associated protein.

PAK1 negatively regulates the activity of the Rho exchange factor NET1.

Rho family small G-protein activity is controlled by guanine nucleotide exchange factors that stimulate the release of GDP, thus allowing GTP binding. Once activated, Rho proteins control cell signaling through interactions with downstream effector proteins, leading to changes in cytoskeletal organization and gene expression. The ability of Rho family members to modulate the activity of other Rho proteins is also intrinsic to these processes. In this work we show that the Rac/Cdc42hs-regulated protein kinase PAK1 down-regulates the activity of the RhoA-specific guanine nucleotide exchange factor NET1. Specifically, PAK1 phosphorylates NET1 on three sites in vitro: serines 152, 153, and 538. Replacement of serines 152 and 153 with glutamate residues down-regulates the activity of NET1 as an exchange factor in vitro and its ability to stimulate actin stress fiber formation in cells. Using a phospho-specific antibody that recognizes NET1 phosphorylated on serine 152, we show that PAK1 phosphorylates NET1 on this site in cells and that Rac1 stimulates serine 152 phosphorylation in a PAK1-dependent manner. Furthermore, coexpression of constitutively active PAK1 inhibits the ability of NET1 to stimulate actin polymerization only when serines 152 and 153 are present. These data provide a novel mechanism for the control of RhoA activity by Rac1 through the PAK-dependent phosphorylation of NET1 to reduce its activity as a guanine nucleotide exchange factor.

Assembly of cytochrome c oxidase within the mitochondrion.

Cytochrome c oxidase (CcO) is an oligomeric complex localized within the mitochondrial inner membrane. Assembly of the active oxidase complex requires the coordinate assembly of subunits synthesized in both the cytoplasm and the mitochondrion. In addition, assembly is dependent on the insertion of five types of cofactors, including two hemes, three copper ions, and one Zn, Mg, and Na ion. A series of accessory proteins are critical for synthesis of the heme A cofactor and insertion of the copper ions. This Account will focus on the steps in the coordinate assembly of CcO subunits, the formation of heme A, and the delivery and insertion of copper ions.

Yeast Cox11, a protein essential for cytochrome c oxidase assembly, is a Cu(I)-binding protein.

Cox11 is a protein essential for respiratory growth and has been implicated in the assembly of the Cu(B) site of cytochrome c oxidase. In the present study, we demonstrate that Cox11 is a copper-binding protein. The soluble C-terminal domain of Cox11 forms a dimer that coordinates one Cu(I) per monomer via three thiolate ligands. The two Cu(I) ions in the dimer exist in a binuclear cluster and appear to be ligated by three conserved Cys residues. Mutation of any of these Cys residues reduces Cu(I) binding and confers respiratory incompetence. Cytochrome c oxidase activity is reduced in these mutants. Thus, the residues important for Cu(I) binding correlate with in vivo function, suggesting that Cu(I) binding is important in Cox11 function.

Activation of soluble guanylyl cyclase by four-coordinate metalloporphyrins: evidence for a role for porphyrin conformation.

Four-coordinate metalloporphyrins activate soluble guanylyl cyclase. Ni(II)PPIX and Cu(II)PPIX are high affinity activators, with activation constants of 24 and 17 nM, respectively. Both metalloporphyrins remain stably bound to the enzyme, enabling spectroscopic characterization of the Ni(II)- and Cu(II)-reconstituted protein. Electronic absorption and resonance Raman spectroscopy reveal that Ni(II)PPIX remains four coordinate when bound to soluble guanylyl cyclase. Analysis of the vibrational frequencies of the Ni(II)-reconstituted enzyme suggests that the protein imposes a constraining force on the porphyrin, favoring a planar conformation. Spectroscopic data for the Cu(II)-substituted protein are also consistent with four coordination. The intensification of the vibrational modes of the peripheral vinyl groups in both Ni(II)- and Cu(II)-reconstituted soluble guanylyl cyclase are consistent with a substantial influence of the protein on the porphyrin environment. Together these data support a model where activation of soluble guanylyl cyclase correlates with the absence of a metal-to-proximal histidine bond and with decreased conformational freedom for the tetrapyrrole in the activated state.