Alexander Trockenbacher

MID1 , mutated in Opitz syndrome, encodes an ubiquitin ligase that targets phosphatase 2A for degradation

The gene MID1, the mutation of which causes X-linked Opitz G/BBB syndrome (OS, MIM 300000), encodes a microtubule-associated protein (MAP). We show that mutation of MID1 leads to a marked accumulation of the catalytic subunit of protein phosphatase 2A (PP2Ac), a central cellular regulator. PP2Ac accumulation is caused by an impairment of a newly identified E3 ubiquitin ligase activity of the MID1 protein that normally targets PP2Ac for degradation through binding to its α4 regulatory subunit in an embryonic fibroblast line derived from a fetus with OS. Elevated PP2Ac causes hypophosphorylation of MAPs, a pathological mechanism that is consistent with the OS phenotype.

Attenuation of cell adhesion in lymphocytes is regulated by CYTIP, a protein which mediates signal complex sequestration

An important theme in molecular cell biology is the regulation of protein recruitment to the plasma membrane. Fundamental biological processes such as proliferation, differentiation or leukocyte functions are initiated and controlled through the reversible binding of signaling proteins to phosphorylated membrane components. This is mediated by specialized interaction modules, such as SH2 and PH domains. Cytohesin‐1 is an intracellular guanine nucleotide exchange factor, which regulates leukocyte adhesion. The activity of cytohesin‐1 is controlled by phospho inositide‐dependent membrane recruitment. An interacting protein was identified, the expression of which is upregulated by cytokines in hematopoietic cells. This molecule, CYTIP, is also recruited to the cell cortex by integrin signaling via its PDZ domain. However, stimulation of Jurkat cells with phorbol ester results in re‐localization of CYTIP to the cytoplasm, and membrane detachment of cytohesin‐1 strictly requires co‐expression of CYTIP. Con sequently, stimulated adhesion of Jurkat cells to intracellular adhesion molecule‐1 is repressed by CYTIP. These findings outline a novel mechanism of signal chain abrogation through sequestration of a limiting component by specific protein–protein interactions.

The Opitz syndrome gene product MID1 assembles a microtubule-associated ribonucleoprotein complex

Opitz BBB/G syndrome (OS) is a heterogenous malformation syndrome mainly characterised by hypertelorism and hypospadias. In addition, patients may present with several other defects of the ventral midline such as cleft lip and palate and congenital heart defects. The syndrome-causing gene encodes the X-linked E3 ubiquitin ligase MID1 that mediates ubiquitin-specific modification and degradation of the catalytic subunit of the translation regulator protein phosphatase 2A (PP2A). Here, we show that the MID1 protein also associates with elongation factor 1α (EF-1α) and several other proteins involved in mRNA transport and translation, including RACK1, Annexin A2, Nucleophosmin and proteins of the small ribosomal subunits. Mutant MID1 proteins as found in OS patients lose the ability to interact with EF-1α. The composition of the MID1 protein complex was determined by several independent methods: (1) yeast two-hybrid screening and (2) immunofluorescence, (3) a biochemical approach involving affinity purification of the complex, (4) co-fractionation in a microtubule assembly assay and (5) immunoprecipitation. Moreover, we show that the cytoskeleton-bound MID1/translation factor complex specifically associates with G- and U-rich RNAs and incorporates MID1 mRNA, thus forming a microtubule-associated ribonucleoprotein (RNP) complex. Our data suggest a novel function of the OS gene product in directing translational control to the cytoskeleton. The dysfunction of this mechanism would lead to malfunction of microtubule-associated protein translation and to the development of OS.

Modulation of Cav1.3 Ca2+ channel gating by Rab3 interacting molecule

Neurotransmitter release and spontaneous action potentials during cochlear inner hair cell (IHC) development depend on the activity of Ca(v)1.3 voltage-gated L-type Ca(2+) channels. Their voltage- and Ca(2+)-dependent inactivation kinetics are slower than in other tissues but the underlying molecular mechanisms are not yet understood. We found that Rab3-interacting molecule-2alpha (RIM2alpha) mRNA is expressed in immature cochlear IHCs and the protein co-localizes with Ca(v)1.3 in the same presynaptic compartment of IHCs. Expression of RIM proteins in tsA-201 cells revealed binding to the beta-subunit of the channel complex and RIM-induced slowing of both Ca(2+)- and voltage-dependent inactivation of Ca(v)1.3 channels. By inhibiting inactivation, RIM induced a non-inactivating current component typical for IHC Ca(v)1.3 currents which should allow these channels to carry a substantial window current during prolonged depolarizations. These data suggest that RIM2 contributes to the stabilization of Ca(v)1.3 gating kinetics in immature IHCs.

Regulation of the MID1 protein function is fine-tuned by a complex pattern of alternative splicing

Clinical features of Opitz BBB/G syndrome are confined to defects of the developing ventral midline, whereas the causative gene, MID1, is ubiquitously expressed. Therefore, a non-redundant physiological function of the MID1 product appears to be developmentally restricted. Here, we report the identification of several alternative MID1 exons in human, mouse and fugu. We show that splice variants of the MID1 gene that are comparable in terms of function occur in the three organisms, suggesting an important role in the regulation of the MID1 protein function. Accordingly, we observed differential MID1 transcript patterns in a tissue-specific manner by Northern blot and RT-PCR. The identified splice variants cause loss-of-function effects via several mechanisms. Some introduce a stop codon followed by a novel poly(A+) tail, leading to the formation of C-terminally truncated proteins. Dominant negative effects through altered binding to the MID1-interacting protein α4 in vitro could be demonstrated in a couple of cases. Others carry premature termination codons without poly(A+) tails. These are degraded by nonsense mediated mRNA decay (NMD). Our data reveal a mechanism conserved in human, mouse and fugu that regulates developmentally restricted MID1 activity and suggest NMD to be critical in the translational regulation of a ubiquitously transcribed mRNA.

RIM modulates CaV1.3 Ca2+ channels

Calcium channel β subunits (CaVβs) are essential cytoplasmic components of voltage-gated calcium channels (VGCCs) affecting their gating and targeting. CaVβs bind with high affinity to the cytoplasmic loop between transmembrane segments I and II of the α1 subunit (loop-I-II). To identify new proteins that modulate VGCCs by interaction with CaVβs we performed a yeast two-hybrid screen using CaVβ2a as bait. Screening of a human fetal brain cDNA library identified a C-terminal fragment of RIM1α (Rab3-interacting molecule) which contains a highly conserved C2B domain as potential interaction partner. To proof the interaction between RIM and CaVβs we developed a protein targeting assay in tsA-201 cells heterologously expressing the loop-I-II of CaV1.3 channels with diverse CaVβ subunits. The CaV1.3-loop-I-II was transported to the plasma membrane and co-targeted all CaVβ subunits indicating that the CaV1.3-loop-I-II and the CaVβ subunits formed a functional complex. The C-terminal fragment of RIM1α or the full-length form of RIM2β exhibited a cytoplasmic distribution but when co-expressed with CaVβs in presence of the CaV1.3-loop-I-II both were co-localized at the plasma membrane. Using qualitative RT-PCR analysis we detected various RIM isoforms in the total organ of Corti and RIM2α in cochlea inner hair cells (IHCs) at an early developmental stage, before the onset of hearing. As RIM is a presynaptic active zone protein involved in Ca2+-induced neurotransmitter release, we asked the question whether the association of RIM with CaV1.3 could account for the slow CaV1.3 current inactivation seen in IHCs. In whole-cell patch-clamp analysis of tsA-201 cells using 15 mM Ca2+ as charge carrier the C2B domain containing fragments of RIM1α and RIM2α caused a significant depolarizing shift of the activation-curve of CaV1.3 (7–12 mV) and slowed the inactivation of both Ca2+ and Ba2+ currents (p < 0.05) albeit to a lesser extent as found in native IHCs. To investigate if a slowly inactivating CaV1.3 spliceform (1b) could contribute to this effect we examined its expression with RT-PCR analysis. However, we did not detect CaV1.3(1b) transcripts in the total organ of Corti at the same developmental stage as we found RIM. Taken together these data showed that indeed RIM modulated CaV1.3 Ca2+ channels. However, we assume that a mixture of diverse proteins and/or CaV1.3 splice variants probably accounts for the slow current inactivation of these channels in native IHCs.

Ubiquitination of a Novel form of α-Synuclein by Parkin

The pathogenesis of PD remains unclear, but the existence of genetic susceptibility factors is well documented (1). Three genes encoding parkin (2), α-synuclein (αS) (3,4) and UCH-L1 (5), respectively, were recently linked to familial forms of PD. Missense mutations in αS and UCH-L 1 cause rare autosomal dominant forms of PD (1). In contrast, mutations of parkin are a common cause of autosomal recessive PD (ARPD) (6, 7). The reported pathologic changes ofparkin-linked ARPD are largely confined to the substantia nigra and locus coeruleus and include loss of monoaminergic neurons, gliosis and a lack of the αS-containing Lewy bodies (LB) that are a hallmark of idiopathic PD (9–12).