N-acetylglucosamine transferase is an integral component of a kinesin-directed mitochondrial trafficking complex

Trafficking kinesin proteins (TRAKs) 1 and 2 are kinesin-associated proteins proposed to function in excitable tissues as adaptors in anterograde trafficking of cargoes including mitochondria. They are known to associate with N-acetylglucosamine transferase and the mitochondrial rho GTPase, Miro. We used confocal imaging, Förster resonance energy transfer and immunoprecipitations to investigate association between TRAKs1/2, N-acetylglucosamine transferase, the prototypic kinesin-1, KIF5C, and Miro. We demonstrate that in COS-7 cells, N-acetylglucosamine transferase, KIF5C and TRAKs1/2 co-distribute. Förster resonance energy transfer was observed between N-acetylglucosamine transferase and TRAKs1/2. Despite co-distributing with KIF5C and immunoprecipitations demonstrating a TRAK1/2, N-acetylglucosamine transferase and KIF5C ternary complex, no Förster resonance energy transfer was detected between N-acetylglucosamine transferase and KIF5C. KIF5C, N-acetylglucosamine transferase, TRAKs1/2 and Miro formed a quaternary complex. The presence of N-acteylglucosamine transferase partially prevented redistribution of mitochondria induced by trafficking proteins 1/2 and KIF5C. TRAK2 was a substrate for N-acetylglucosamine transferase with TRAK2 (S562) identified as a site of O-N-acetylglucosamine modification. These findings substantiate trafficking kinesin proteins as scaffolds for the formation of a multi-component complex involved in anterograde trafficking of mitochondria. They further suggest that O-glycosylation may regulate complex formation.     

Ca-dependent binding of calcium-binding protein 1 to presynaptic group III metabotropic glutamate receptors and blockage by phosphorylation of the receptors

Presynaptic group III metabotropic glutamate receptors (mGluRs) and Ca²⁺ channels are the main neuronal activity-dependent regulators of synaptic vesicle release, and they use common molecules in their signaling cascades. Among these, calmodulin (CaM) and the related EF-hand Ca²⁺-binding proteins are of particular importance as sensors of presynaptic Ca²⁺, and a multiple of them are indeed utilized in the signaling of Ca²⁺ channels. However, despite its conserved structure, CaM is the only known EF-hand Ca²⁺-binding protein for signaling by presynaptic group III mGluRs. Because the mGluRs and Ca²⁺ channels reciprocally regulate each other and functionally converge on the regulation of synaptic vesicle release, the mGluRs would be expected to utilize more EF-hand Ca²⁺-binding proteins in their signaling. Here I show that calcium-binding protein 1 (CaBP1) bound to presynaptic group III mGluRs competitively with CaM in a Ca²⁺-dependent manner and that this binding was blocked by protein kinase C (PKC)-mediated phosphorylation of these receptors. As previously shown for CaM, these results indicate the importance of CaBP1 in signal cross talk at presynaptic group III mGluRs, which includes many molecules such as cAMP, Ca²⁺, PKC, G protein, and Munc18-1. However, because the functional diversity of EF-hand calcium-binding proteins is extraordinary, as exemplified by the regulation of Ca²⁺ channels, CaBP1 would provide a distinct way by which presynaptic group III mGluRs fine-tune synaptic transmission.     

Rab11a and its binding partners regulate the recycling of the ß1-adrenergic receptor

ß1-adrenergic receptors (ß1-AR) are internalized in response to agonists and then recycle back for another round of signaling. The serine 312 to alanine mutant of the ß1-AR (S312A) is internalized but does not recycle. We determined that WT ß1-AR and S312A were internalized initially to an early sorting compartment because they colocalized by > 70% with the early endosomal markers rab5a and early endosomal antigen-1 (EEA1). Subsequently, the WT ß1-AR trafficked via rab4a-expressing sorting endosomes to recycling endosomes. In recycling endosomes WT ß1-AR were colocalized by > 70% with the rab11 GTPase. S312A did not colocalize with either rab4a or rab11, instead they exited from early endosomes to late endosomes/lysosomes in which they were degraded. Rab11a played a prominent role in recycling of the WT ß1-AR because dominant negative rab11a inhibited, while constitutively active rab11a accelerated the recycling of the ß1-AR. Next, we determined the effect of each of the rab11-interacting proteins on trafficking of the WT ß1-AR. The recycling of the ß1-AR was markedly inhibited when myosin Vb, FIP2, FIP3 and rabphillin were knocked down. These data indicate that rab11a and a select group of its binding partners play a prominent role in recycling of the human ß1-AR.     

HSP90 is required for TAK1 stability but not for its activation in the pro-inflammatory signaling pathway

The protein kinase transforming-growth-factor-β-activated kinase-1 (TAK1) is a key regulator in the pro-inflammatory signaling pathway and is activated by tumor necrosis factor-α, interleukin-1 (IL-1) and lipopolysaccharide (LPS). We describe the identification of TAK1 as a client protein of the 90 kDa heat-shock protein (Hsp90)/cell division cycle protein 37 (Cdc37) chaperones. However, Hsp90 is not required for the activation of TAK1 as short exposure to the Hsp90 inhibitor, 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) did not affect its activation by LPS or IL-1. Prolonged treatment of cells with 17-AAG inhibits Hsp90 and downregulates TAK1. Our results suggest that Hsp90 is required for the folding and stability of TAK1 but is displaced and no longer required when TAK1 is complexed to TAK1-binding protein-1 (TAB1).

Structured summary

MINT-6797182:

TAK1 (uniprotkb:O43318-2) physically interacts (MI:0218) with CDC37 (uniprotkb:Q16543) and HSP90 (uniprotkb:P07900) by anti bait coimmunoprecipitation (MI:0006)

MINT-6797194:

TAK1 (uniprotkb:O43318-2) physically interacts (MI:0218) with TAB1 (uniprotkb:Q15750), HSP90 (uniprotkb:P07900) and CDC37 (uniprotkb:Q16543) by anti bait coimmunoprecipitation (MI:0006)

MINT-6797248:

TAK1 (uniprotkb:Q62073) physically interacts (MI:0218) with HSP90 (uniprotkb:P07901), CDC37 (uniprotkb:Q61081), TAB2 (uniprotkb:Q99K90) and TAB1 (uniprotkb:Q8CF89) by anti bait coimmunoprecipitation (MI:0006)

MINT-6797232:

TAK1 (uniprotkb:O43318-2) physically interacts (MI:0218) with HSP90 (uniprotkb:P07900) and CDC37 (uniprotkb:Q16543) by pull down (MI:0096)

MINT-6797216:

TAK1 (uniprotkb:O43318-2) physically interacts (MI:0218) with TAB2 (uniprotkb:Q9NYJ8), CDC37 (uniprotkb:Q16543), HSP90 (uniprotkb:P07900) and TAB1 (uniprotkb:Q15750) by anti bait coimmunoprecipitation (MI:0006)

     

Modulation of the K(v)4.3 channel by syntaxin 1A

The SNARE protein syntaxin 1A (Syn1A) is known to inhibit delayed rectifier K(+) channels of the K(v)1 and K(v)2 families with heterogeneous effects on their gating properties. In this study, we explored whether Syn1A could directly modulate K(v)4.3, a rapidly inactivating K(v) channel with important roles in neuroendocrine cells and cardiac myocytes. Immunoprecipitation studies in HEK293 cells coexpressing Syn1A and K(v)4.3 revealed a direct interaction with increased trafficking to the plasma membrane without a change in channel synthesis. Paradoxically, Syn1A inhibited K(v)4.3 current density. In particular, Syn1A produced a left-shift in steady-state inactivation of K(v)4.3 without affecting either voltage dependence of activation or gating kinetics, a pattern distinct from other K(v) channels. Combined with our previous reports, our results further verify the notion that the mechanisms involved in Syn1A-K(v) interactions vary significantly between K(v) channels, thus providing a wide scope for Syn1A modulation of exocytosis and membrane excitability.