Sumo Modification of Ion Channels.

Recently, a role for SUMO modification outside of the nucleus has emerged. Although the number of extranuclear proteins known to be sumoylated is comparatively small, ion channels represent one important new class of these proteins. Ion channels are responsible for the control of membrane excitability and therefore are critical for fundamental physiological processes such as muscle contraction, neuronal firing, and cellular homeostasis. As such, these ion-conducting proteins are subject to precise regulation. Recently, several studies have identified sumoylation as a novel mechanism of modulating ion channel function. These studies expand the list of known functions of sumoylation and reveal that, in addition to its more established role in the regulation of nuclear proteins, this modification plays important roles at the cytoplasmic face of membranes.

SUMOylation regulates ciliary localization of olfactory signaling proteins.

Cilia are evolutionarily conserved organelles found on many mammalian cell types, including neuronal populations. Although neuronal cilia, including those on olfactory sensory neurons (OSNs), are often delineated by localization of adenylyl cyclase 3 (AC3, also known as ADCY3), the mechanisms responsible for targeting integral membrane proteins are largely unknown. Post-translational modification by small ubiquitin-like modifier (SUMO) proteins plays an important role in protein localization processes such as nuclear-cytosolic transport. Here, we identified through bioinformatic analysis that adenylyl cyclases harbor conserved SUMOylation motifs, and show that AC3 is a substrate for SUMO modification. Functionally, overexpression of the SUMO protease SENP2 prevented ciliary localization of AC3, without affecting ciliation or cilia maintenance. Furthermore, AC3-SUMO mutants did not localize to cilia. To test whether SUMOylation is sufficient for cilia entry, we compared localization of ANO2, which possesses a SUMO motif, and ANO1, which lacks SUMOylation sites and does not localize to cilia. Introduction of SUMOylation sites into ANO1 was not sufficient for ciliary entry. These data suggest that SUMOylation is necessary but not sufficient for ciliary trafficking of select constituents, further establishing the link between ciliary and nuclear import.

Disrupting SUMOylation enhances transcriptional function and ameliorates polyglutamine androgen receptor-mediated disease.

Expansion of the polyglutamine (polyQ) tract within the androgen receptor (AR) causes neuromuscular degeneration in individuals with spinobulbar muscular atrophy (SBMA). PolyQ AR has diminished transcriptional function and exhibits ligand-dependent proteotoxicity, features that have both been implicated in SBMA; however, the extent to which altered AR transcriptional function contributes to pathogenesis remains controversial. Here, we sought to dissociate effects of diminished AR function from polyQ-mediated proteotoxicity by enhancing the transcriptional activity of polyQ AR. To accomplish this, we bypassed the inhibitory effect of AR SUMOylation (where SUMO indicates small ubiquitin-like modifier) by mutating conserved lysines in the polyQ AR that are sites of SUMOylation. We determined that replacement of these residues by arginine enhances polyQ AR activity as a hormone-dependent transcriptional regulator. In a murine model, disruption of polyQ AR SUMOylation rescued exercise endurance and type I muscle fiber atrophy; it also prolonged survival. These changes occurred without overt alterations in polyQ AR expression or aggregation, revealing the favorable trophic support exerted by the ligand-activated receptor. Our findings demonstrate beneficial effects of enhancing the transcriptional function of the ligand-activated polyQ AR and indicate that the SUMOylation pathway may be a potential target for therapeutic intervention in SBMA.

Bifunctional ligands allow deliberate extrinsic reprogramming of the glucocorticoid receptor.

Therapies based on conventional nuclear receptor ligands are extremely powerful, yet their broad and long-term use is often hindered by undesired side effects that are often part of the receptor's biological function. Selective control of nuclear receptors such as the glucocorticoid receptor (GR) using conventional ligands has proven particularly challenging. Because they act solely in an allosteric manner, conventional ligands are constrained to act via cofactors that can intrinsically partner with the receptor. Furthermore, effective means to rationally encode a bias for specific coregulators are generally lacking. Using the (GR) as a framework, we demonstrate here a versatile approach, based on bifunctional ligands, that extends the regulatory repertoire of GR in a deliberate and controlled manner. By linking the macrolide FK506 to a conventional agonist (dexamethasone) or antagonist (RU-486), we demonstrate that it is possible to bridge the intact receptor to either positively or negatively acting coregulatory proteins bearing an FK506 binding protein domain. Using this strategy, we show that extrinsic recruitment of a strong activation function can enhance the efficacy of the full agonist dexamethasone and reverse the antagonist character of RU-486 at an endogenous locus. Notably, the extrinsic recruitment of histone deacetylase-1 reduces the ability of GR to activate transcription from a canonical GR response element while preserving ligand-mediated repression of nuclear factor-κB. By providing novel ways for the receptor to engage specific coregulators, this unique ligand design approach has the potential to yield both novel tools for GR study and more selective therapeutics.

RSUME enhances glucocorticoid receptor SUMOylation and transcriptional activity.

Glucocorticoid receptor (GR) activity is modulated by posttranslational modifications, including phosphorylation, ubiquitination, and SUMOylation. The GR has three SUMOylation sites: lysine 297 (K297) and K313 in the N-terminal domain (NTD) and K721 within the ligand-binding domain. SUMOylation of the NTD sites mediates the negative effect of the synergy control motifs of GR on promoters with closely spaced GR binding sites. There is scarce evidence on the role of SUMO conjugation to K721 and its impact on GR transcriptional activity. We have previously shown that RSUME (RWD-containing SUMOylation enhancer) increases protein SUMOylation. We now demonstrate that RSUME interacts with the GR and increases its SUMOylation. RSUME regulates GR transcriptional activity and the expression of its endogenous target genes, FKBP51 and S100P. RSUME uncovers a positive role for the third SUMOylation site, K721, on GR-mediated transcription, demonstrating that GR SUMOylation acts positively in the presence of a SUMOylation enhancer. Both mutation of K721 and small interfering RNA-mediated RSUME knockdown diminish GRIP1 coactivator activity. RSUME, whose expression is induced under stress conditions, is a key factor in heat shock-induced GR SUMOylation. These results show that inhibitory and stimulatory SUMO sites are present in the GR and at higher SUMOylation levels the stimulatory one becomes dominant.

The in vivo role of androgen receptor SUMOylation as revealed by androgen insensitivity syndrome and prostate cancer mutations targeting the proline/glycine residues of synergy control motifs.

The androgen receptor (AR) mediates the effects of male sexual hormones on development and physiology. Alterations in AR function are central to reproductive disorders, prostate cancer, and Kennedy disease. AR activity is influenced by post-translational modifications, but their role in AR-based diseases is poorly understood. Conjugation by small ubiquitin-like modifier (SUMO) proteins at two synergy control (SC) motifs in AR exerts a promoter context-dependent inhibitory role. SC motifs are composed of a four-amino acid core that is often preceded and/or followed by nearby proline or glycine residues. The function of these flanking residues, however, has not been examined directly. Remarkably, several AR mutations associated with oligospermia and androgen insensitivity syndrome map to Pro-390, the conserved proline downstream of the first SC motif in AR. Similarly, mutations at Gly-524, downstream of the second SC motif, were recovered in recurrent prostate cancer samples. We now provide evidence that these clinically isolated substitutions lead to a partial loss of SC motif function and AR SUMOylation that affects multiple endogenous genes. Consistent with a structural role as terminators of secondary structure elements, substitution of Pro-390 by Gly fully supports both SC motif function and SUMOylation. As predicted from the functional properties of SC motifs, the clinically isolated mutations preferentially enhance transcription driven by genomic regions harboring multiple AR binding sites. The data support the view that alterations in AR SUMOylation play significant roles in AR-based diseases and offer novel SUMO-based therapeutic opportunities.

Small ubiquitin-like modifier (SUMO) modification mediates function of the inhibitory domains of developmental regulators FOXC1 and FOXC2.

FOXC1 and FOXC2 are forkhead transcription factors that play essential roles during development and physiology. Despite their critical role, the mechanisms that regulate the function of these factors remain poorly understood. We have identified conserved motifs within a previously defined N-terminal negative regulatory region of FOXC1/C2 that conforms to the definition of synergy control or SC motifs. Because such motifs inhibit the activity of transcription factors by serving as sites of post-translational modification by small ubiquitin-like modifier (SUMO), we have examined whether FOXC1/C2 are targets of SUMOylation and probed the functional significance of this modification. We find that endogenous FOXC1 forms modified by SUMO2/3 can be detected. Moreover, in cell culture, all three SUMO isoforms are readily conjugated to FOXC1 and FOXC2. The modification can be reconstituted in vitro with purified components and can be reversed in vitro by treatment with the SUMO protease SENP2. SUMOylation of FOXC1 and FOXC2 occurs primarily on one consensus synergy control motif with minor contributions of a second, more degenerate site. Notably, although FOXC1 is also phosphorylated at multiple sites, disruption of sites immediately downstream of the SC motifs does not influence SUMOylation. Consistent with a negative functional role, SUMOylation-deficient mutants displayed higher transcriptional activity when compared with wild type forms despite comparable protein levels and subcellular localization. Thus, the findings demonstrate that SC motifs mediate the inhibitory function of this region by serving as sites for SUMOylation and reveal a novel mechanism for acute and reversible regulation of FOXC1/C2 function.

Subunit-dependent axonal trafficking of distinct alpha heteromeric potassium channel complexes.

Voltage-gated potassium (Kv) channels are critical for neuronal excitability and are targeted to specific subcellular compartments to carry out their unique functions. While it is widely believed that Kv channels exist as heteromeric complexes in neurons, direct tests of the hypothesis that specific heteromeric channel populations display divergent spatial and temporal dynamics are limited. Using a bimolecular fluorescence complementation approach, we monitored the assembly and localization of cell surface channel complexes in living cells. While PSD95-mediated clustering was subunit independent, selective visualization of heteromeric Kv complexes in rat hippocampal neurons revealed subunit-dependent localization that was not predicted by analyzing individual subunits. Assembly of Kv1.1 with Kv1.4 prevented axonal localization but not surface expression, while inclusion of Kv1.2 imparted clustering at presynaptic sites and decreased channel mobility within the axon. This mechanism by which specific Kv channel subunits can act in a dominant manner to impose unique trafficking properties to heteromeric complexes extended to Shab-related family of Kv channels. When coexpressed, Kv2.1 and Kv2.2 heteromultimers did not aggregate in somatodendritic clusters observed with expression of Kv2.1 alone. These studies demonstrate selective axonal trafficking and surface localization of distinct Kv channels based on their subunit composition.

Keratin hypersumoylation alters filament dynamics and is a marker for human liver disease and keratin mutation.

Keratin polypeptide 8 (K8) associates noncovalently with its partners K18 and/or K19 to form the intermediate filament cytoskeleton of hepatocytes and other simple-type epithelial cells. Human K8, K18, and K19 variants predispose to liver disease, whereas site-specific keratin phosphorylation confers hepatoprotection. Because stress-induced protein phosphorylation regulates sumoylation, we hypothesized that keratins are sumoylated in an injury-dependent manner and that keratin sumoylation is an important regulatory modification. We demonstrate that K8/K18/K19, epidermal keratins, and vimentin are sumoylated in vitro. Upon transfection, K8, K18, and K19 are modified by poly-SUMO-2/3 chains on Lys-285/Lys-364 (K8), Lys-207/Lys-372 (K18), and Lys-208 (K19). Sumoylation affects filament organization and stimulus-induced keratin solubility and is partially inhibited upon mutation of one of three known K8 phosphorylation sites. Extensive sumoylation occurs in cells transfected with individual K8, K18, or K19 but is limited upon heterodimerization (K8/K18 or K8/K19) in the absence of stress. In contrast, keratin sumoylation is significantly augmented in cells and tissues during apoptosis, oxidative stress, and phosphatase inhibition. Poly-SUMO-2/3 conjugates are present in chronically injured but not normal, human, and mouse livers along with polyubiquitinated and large insoluble keratin-containing complexes. Notably, common human K8 liver disease-associated variants trigger keratin hypersumoylation with consequent diminished solubility. In contrast, modest sumoylation of wild type K8 promotes solubility. Hence, conformational changes induced by keratin natural mutations and extensive tissue injury result in K8/K18/K19 hypersumoylation, which retains keratins in an insoluble compartment, thereby limiting their cytoprotective function.

SUMOylation of the mitochondrial fission protein Drp1 occurs at multiple nonconsensus sites within the B domain and is linked to its activity cycle.

Dynamin-related protein (Drp) 1 is a key regulator of mitochondrial fission and is composed of GTP-binding, Middle, insert B, and C-terminal GTPase effector (GED) domains. Drp1 associates with mitochondrial fission sites and promotes membrane constriction through its intrinsic GTPase activity. The mechanisms that regulate Drp1 activity remain poorly understood but are likely to involve reversible post-translational modifications, such as conjugation of small ubiquitin-like modifier (SUMO) proteins. Through a detailed analysis, we find that Drp1 interacts with the SUMO-conjugating enzyme Ubc9 via multiple regions and demonstrate that Drp1 is a direct target of SUMO modification by all three SUMO isoforms. While Drp1 does not harbor consensus SUMOylation sequences, our analysis identified2 clusters of lysine residues within the B domain that serve as noncanonical conjugation sites. Although initial analysis indicates that mitochondrial recruitment of ectopically expressed Drp1 in response to staurosporine is unaffected by loss of SUMOylation, we find that Drp1 SUMOylation is enhanced in the context of the K38A mutation. This dominant-negative mutant, which is deficient in GTP binding and hydrolysis, does not associate with mitochondria and prevents normal mitochondrial fission. This finding suggests that SUMOylation of Drp1 is linked to its activity cycle and is influenced by Drp1 localization.

Small ubiquitin-like modifier (SUMO) modification of the androgen receptor attenuates polyglutamine-mediated aggregation.

The neurodegenerative disorder spinal and bulbar muscular atrophy or Kennedy disease is caused by a CAG trinucleotide repeat expansion within the androgen receptor (AR) gene. The resulting expanded polyglutamine tract in the N-terminal region of the receptor renders AR prone to ligand-dependent misfolding and formation of oligomers and aggregates that are linked to neuronal toxicity. How AR misfolding is influenced by post-translational modifications, however, is poorly understood. AR is a target of SUMOylation, and this modification inhibits AR activity in a promoter context-dependent manner. SUMOylation is up-regulated in response to multiple forms of cellular stress and may therefore play an important cytoprotective role. Consistent with this view, we find that gratuitous enhancement of overall SUMOylation significantly reduced the formation of polyglutamine-expanded AR aggregates without affecting the levels of the receptor. Remarkably, this effect requires SUMOylation of AR itself because it depends on intact AR SUMOylation sites. Functional analyses, however, indicate that the protective effects of enhanced AR SUMOylation are not due to alterations in AR transcriptional activity because a branched protein structure in the appropriate context of the N-terminal region of AR is necessary to antagonize aggregation but not for inhibiting AR transactivation. Remarkably, small ubiquitin-like modifier (SUMO) attenuates AR aggregation through a unique mechanism that does not depend on critical features essential for its interaction with canonical SUMO binding motifs. Our findings therefore reveal a novel function of SUMOylation and suggest that approaches that enhance AR SUMOylation may be of clinical use in polyglutamine expansion diseases.

SUMOylation inhibits SF-1 activity by reducing CDK7-mediated serine 203 phosphorylation.

Steroidogenic factor 1 (SF-1) is an orphan nuclear receptor selectively expressed in the adrenal cortex and gonads, where it mediates the hormonal stimulation of multiple genes involved in steroid hormone biosynthesis. SF-1 is the target of both phosphorylation and SUMOylation, but how these modifications interact or contribute to SF-1 regulation of endogenous genes remains poorly defined. We found that SF-1 is selectively SUMOylated at K194 in Y1 adrenocarcinoma cells and that although SUMOylation does not alter the subcellular localization of SF-1, the modification inhibits the ability of SF-1 to activate target genes. Notably, whereas SF-1 SUMOylation is independent of S203 phosphorylation and is unaffected by adrenocorticotropin (ACTH) treatment, loss of SUMOylation leads to enhanced SF-1 phosphorylation at serine 203. Furthermore, preventing SF-1 SUMOylation increases the mRNA and protein levels of multiple steroidogenic enzyme genes. Analysis of the StAR promoter indicates that blockade of SF-1 SUMOylation leads to an increase in overall promoter occupancy but does not alter the oscillatory recruitment dynamics in response to ACTH. Notably, we find that CDK7 binds preferentially to the SUMOylation-deficient form of SF-1 and that CDK7 inhibition reduces phosphorylation of SF-1. Based on these observations, we propose a coordinated modification model in which inhibition of SF-1-mediated transcription by SUMOylation in adrenocortical cancer cells is mediated through reduced CDK7-induced phosphorylation of SF-1.

SUMO-mediated inhibition of glucocorticoid receptor synergistic activity depends on stable assembly at the promoter but not on DAXX.

Multiple transcription factors, including members of the nuclear receptor family, harbor one or more copies of a short regulatory motif that limits synergistic transactivation in a context-dependent manner. These synergy control (SC) motifs exert their effects by serving as sites for posttranslational modification by small ubiquitin-like modifier (SUMO) proteins. By analyzing the requirements for both synergy control and SUMOylation in the glucocorticoid receptor (GR), we find that an intact ligand-binding domain and an engaged DNA- binding domain dimerization interface are necessary for effective synergy control. However, these features, which promote stable assembly of GR-DNA complexes, are required downstream of SUMOylation because their disruption or deletion does not interfere with SUMO modification. Remarkably, in the absence of these features, sensitivity to the effects of SUMOylation can be restored simply by stabilization of DNA interactions through a heterologous DNA binding domain. The data indicate that stable interaction with DNA is an important prerequisite for SUMO-dependent transcriptional inhibition. Analysis of genomic regions occupied by GR indicates that the effects of SC motif SUMOylation are most evident at multiple, near-ideal GR binding sites and that SUMOylation selectively affects the induction of linked endogenous genes. Although the SUMO-binding protein DAXX has been proposed to mediate the inhibitory effects of GR SUMOylation, we find that inhibition by DAXX is independent of GR SUMOylation. Furthermore, neither expression nor knockdown of DAXX influences SUMO effects on GR. We therefore propose that stable binding of GR to multiple sites on DNA allows for the SUMO-dependent recruitment of inhibitory factors distinct from DAXX.

Acetylation and deacetylation regulate CCAAT/enhancer binding protein beta at K39 in mediating gene transcription.

The transcription factor CCAAT/enhancer binding protein beta (C/EBPbeta) contains multiple acetylation sites, including lysine (K) 39. Mutation of C/EBPbeta at K39, an acetylation site in the transcriptional activation domain, impairs transcription of C/EBPbeta target genes in a dominant-negative fashion. Further, K39 of C/EBPbeta can be deacetylated by HDAC1, and HDAC1 may decrease C/EBPbeta-mediated transcription, suggesting that acetylation of C/EBPbeta at K39 is dynamically regulated in mediating gene transcription. Acetylation of endogenous C/EBPbeta at K39 is detected in adipose tissue, and also occurs in 3T3-L1 cells undergoing adipocyte conversion. In addition, mutation of K39 in C/EBPbeta impairs activation of its target genes encoding C/EBPalpha and PPARgamma, essential mediators of adipogenesis, as well as adipocyte genes for leptin and Glut4. These findings suggest that acetylation of C/EBPbeta at K39 is an important and dynamic regulatory event that contributes to its ability to transactivate target genes, including those associated with adipogenesis and adipocyte function.

Sumoylation-dependent control of homotypic and heterotypic synergy by the Kruppel-type zinc finger protein ZBP-89.

The Krüppel-like transcription factor ZBP-89 is a sequence-specific regulator that plays key roles in cellular growth and differentiation especially in endodermal and germ cell lineages. ZBP-89 shares with other members of the Sp-like family an overlapping sequence specificity for GC-rich sequences in the regulatory regions of multiple genes. Defining the mechanisms that govern the intrinsic function of ZBP-89 as well as its competitive and non-competitive functional interactions with other regulators is central to understand how ZBP-89 exerts its biological functions. We now describe that post-translational modification of ZBP-89 by multiple small ubiquitin-like modifier (SUMO) isoforms occurs at two conserved synergy control motifs flanking the DNA binding domain. Functionally sumoylation did not directly alter the ability of ZBP-89 to compete with other Sp-like factors from individual sites. At promoters bearing multiple response elements, however, this modification inhibited the functional cooperation between ZBP-89 and Sp1. Analysis of the properties of ZBP-89 in cellular contexts devoid of competing factors indicated that although on its own it behaves as a modest activator it potently synergizes with heterologous activators such as the glucocorticoid receptor. Notably we found that when conjugated to ZBP-89, SUMO exerts a strong inhibitory effect on such synergistic interactions through a critical conserved functional surface. By regulating higher order functional interactions, sumoylation provides a reversible post-translational mechanism to control the activity of ZBP-89.

CCG-1423: a small-molecule inhibitor of RhoA transcriptional signaling.

Lysophosphatidic acid receptors stimulate a Galpha(12/13)/RhoA-dependent gene transcription program involving the serum response factor (SRF) and its coactivator and oncogene, megakaryoblastic leukemia 1 (MKL1). Inhibitors of this pathway could serve as useful biological probes and potential cancer therapeutic agents. Through a transcription-based high-throughput serum response element-luciferase screening assay, we identified two small-molecule inhibitors of this pathway. Mechanistic studies on the more potent CCG-1423 show that it acts downstream of Rho because it blocks SRE.L-driven transcription stimulated by Galpha(12)Q231L, Galpha(13)Q226L, RhoA-G14V, and RhoC-G14V. The ability of CCG-1423 to block transcription activated by MKL1, but not that induced by SRF-VP16 or GAL4-VP16, suggests a mechanism targeting MKL/SRF-dependent transcriptional activation that does not involve alterations in DNA binding. Consistent with its role as a Rho/SRF pathway inhibitor, CCG-1423 displays activity in several in vitro cancer cell functional assays. CCG-1423 potently (<1 mumol/L) inhibits lysophosphatidic acid-induced DNA synthesis in PC-3 prostate cancer cells, and whereas it inhibits the growth of RhoC-overexpressing melanoma lines (A375M2 and SK-Mel-147) at nanomolar concentrations, it is less active on related lines (A375 and SK-Mel-28) that express lower levels of Rho. Similarly, CCG-1423 selectively stimulates apoptosis of the metastasis-prone, RhoC-overexpressing melanoma cell line (A375M2) compared with the parental cell line (A375). CCG-1423 inhibited Rho-dependent invasion by PC-3 prostate cancer cells, whereas it did not affect the Galpha(i)-dependent invasion by the SKOV-3 ovarian cancer cell line. Thus, based on its profile, CCG-1423 is a promising lead compound for the development of novel pharmacologic tools to disrupt transcriptional responses of the Rho pathway in cancer.

Rab-GTPase-dependent endocytic recycling of Kv1.5 in atrial myocytes.

The number of ion channels expressed on the cell surface shapes the complex electrical response of excitable cells. Maintaining a balance between anterograde and retrograde trafficking of channel proteins is vital in regulating steady-state cell surface expression. Kv1.5 is an important voltage-gated K(+) channel in the cardiovascular system underlying the ultra-rapid rectifying potassium current (Ik(ur)), a major repolarizing current in atrial myocytes, and regulating the resting membrane potential and excitability of smooth muscle cells. Defects in the expression of Kv1.5 are associated with pathological states such as chronic atrial fibrillation and hypoxic pulmonary hypertension. There is, thus, substantial interest in understanding the mechanisms regulating cell surface channel levels. Here, we investigated the internalization and recycling of Kv1.5 in the HL-1 immortalized mouse atrial myocytes. Kinetic studies indicate that Kv1.5 is rapidly internalized to a perinuclear region where it co-localizes with the early endosomal marker, EEA1. Importantly, we identified that a population of Kv1.5, originating on the cell surface, internalized and recycled back to the plasma membrane. Notably, Kv1.5 recycling processes are driven by specific Rab-dependent endosomal compartments. Thus, co-expression of GDP-locked Rab4S22N and Rab11S25N dominant-negative mutants decreased the steady-state Kv1.5 surface levels, whereas GTPase-deficient Rab4Q67L and Rab11Q70L mutants increased steady-state Kv1.5 surface levels. These data reveal an unexpected dynamic trafficking of Kv1.5 at the myocyte plasma membrane and demonstrate a role for recycling in the maintenance of steady-state ion channel surface levels.

SUMO modification regulates inactivation of the voltage-gated potassium channel Kv1.5.

The voltage-gated potassium (Kv) channel Kv1.5 mediates the I(Kur) repolarizing current in human atrial myocytes and regulates vascular tone in multiple peripheral vascular beds. Understanding the complex regulation of Kv1.5 function is of substantial interest because it represents a promising pharmacological target for the treatment of atrial fibrillation and hypoxic pulmonary hypertension. Herein we demonstrate that posttranslational modification of Kv1.5 by small ubiquitin-like modifier (SUMO) proteins modulates Kv1.5 function. We have identified two membrane-proximal and highly conserved cytoplasmic sequences in Kv1.5 that conform to established SUMO modification sites in transcription factors. We find that Kv1.5 interacts specifically with the SUMO-conjugating enzyme Ubc9 and is a target for modification by SUMO-1, -2, and -3 in vivo. In addition, purified recombinant Kv1.5 serves as a substrate in a minimal in vitro reconstituted SUMOylation reaction. The SUMO-specific proteases SENP2 and Ulp1 efficiently deconjugate SUMO from Kv1.5 in vivo and in vitro, and disruption of the two identified target motifs results in a loss of the major SUMO-conjugated forms of Kv1.5. In whole-cell patch-clamp electrophysiological studies, loss of Kv1.5 SUMOylation, by either disruption of the conjugation sites or expression of the SUMO protease SENP2, leads to a selective approximately 15-mV hyperpolarizing shift in the voltage dependence of steady-state inactivation. Reversible control of voltage-sensitive channels through SUMOylation constitutes a unique and likely widespread mechanism for adaptive tuning of the electrical excitability of cells.

Multiple mechanisms of growth hormone-regulated gene transcription.

Diverse physiological actions of growth hormone (GH) are mediated by changes in gene transcription. Transcription can be regulated at several levels, including post-translational modification of transcription factors, and formation of multiprotein complexes involving transcription factors, co-regulators and additional nuclear proteins; these serve as targets for regulation by hormones and signaling pathways. Evidence that GH regulates transcription at multiple levels is exemplified by analysis of the proto-oncogene c-fos. Among the GH-regulated transcription factors on c-fos, C/EBPbeta appears to be key, since depletion of C/EBPbeta by RNA interference blocks the stimulation of c-fos by GH. The phosphorylation state of C/EBPbeta and its ability to activate transcription are regulated by GH through MAPK and PI3K/Akt-mediated signaling cascades. The acetylation of C/EBPbeta also contributes to its ability to activate c-fos transcription. These and other post-translational modifications of C/EBPbeta appear to be integrated for regulation of transcription by GH. The formation of nuclear proteins into complexes associated with DNA-bound transcription factors is also regulated by GH. Both C/EBPbeta and the co-activator p300 are recruited to c-fos in response to GH, altering c-fos promoter activation. In addition, GH rapidly induces spatio-temporal re-localization of C/EBPbeta within the nucleus. Thus, GH-regulated gene transcription mediated by C/EBPbeta reflects the integration of diverse mechanisms including post-translational modifications, modulation of protein complexes associated with DNA and re-localization of gene regulatory proteins. Similar integration involving other transcription factors, including Stats, appears to be a feature of regulation by GH of other gene targets.