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.

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.

To develop a genetically faithful model of medulloblastoma with increased tumor incidence compared with the current best model we activated the Sonic Hedgehog (Shh) pathway by transgenically expressing a constitutively active form of Smoothened in mouse cerebellar granule neuron precursors (ND2:SmoA1 mice). This resulted in early cerebellar granule cell hyper-proliferation and a 48% incidence of medulloblastoma formation. Gene expression studies showed an increase in the known Shh targets Gli1 and Nmyc that correlated with increasing hyperplasia and tumor formation. Notch2 and the Notch target gene, HES5, were also significantly elevated in Smoothened-induced tumors showing that Shh pathway activation is sufficient to induce Notch pathway signaling. In human medulloblastomas reverse transcription-PCR for Shh and Notch targets revealed activation of both of these pathways in most tumors when compared with normal cerebellum. Notch pathway inhibition with soluble Delta ligand or gamma secretase inhibitors resulted in a marked reduction of viable cell numbers in medulloblastoma cell lines and primary tumor cultures. Treatment of mice with D283 medulloblastoma xenografts with a gamma secretase inhibitor resulted in decreased proliferation and increased apoptosis, confirming that Notch signaling contributes to human medulloblastoma proliferation and survival. Medulloblastomas in ND2:SmoA1 mice and humans have concomitant increase in Shh and Notch pathway activities, both of which contribute to tumor survival.

One of the most common forms of functional interaction among transcription factors is the more than additive effect at promoters harboring multiple copies of a response element. The mechanisms that enable or control synergy at such compound response elements are poorly understood. We recently defined a common motif within the negative regulatory regions of multiple factors that operates by regulating their transcriptional synergy. We have identified such a synergy control (SC) motif embedded within the "attenuator domain" of CCAAT/enhancer-binding protein alpha (C/EBPalpha), a key regulator of energy homeostasis and cellular differentiation. A Lys(159) –> Arg substitution within the SC motif does not alter C/EBPalpha activity from a single site but leads to enhanced transactivation from synthetic or natural compound response elements. The sequence of SC motifs overlaps with the recently defined consensus SUMO modification site, and we find that the SC motif is the major site of both SUMO-1 and SUMO-3 modification in C/EBPalpha. Furthermore, the disruption of SC motif function is accompanied by loss of SUMO but not ubiquitin modification. C/EBPalpha interacts directly with the E2 SUMO-conjugating enzyme Ubc9 and can be SUMOylated in vitro using purified recombinant components. Notably, we find that PIASy has E3-like activity and enhances both SUMO-1 and SUMO-3 modification of C/EBPalpha in vivo and in vitro. Our results indicate that SUMO modification of SC motifs provides a means to rapidly control higher order interactions among transcription factors and suggests that SUMOylation may be a general mechanism to limit transcriptional synergy.