Abstract
Inositol pyrophosphates, such as diphosphoinositol pentakisphosphate (IP7), are conserved eukaryotic signaling molecules that possess pyrophosphate and monophosphate moieties. Generated predominantly by inositol hexakisphosphate kinases (IP6Ks), inositol pyrophosphates can modulate protein function by posttranslational serine pyrophosphorylation. Here, we report inositol pyrophosphates as novel regulators of cytoplasmic dynein-driven vesicle transport. Mammalian cells lacking IP6K1 display defects in dynein-dependent trafficking pathways, including endosomal sorting, vesicle movement, and Golgi maintenance. Expression of catalytically active but not inactive IP6K1 reverses these defects, suggesting a role for inositol pyrophosphates in these processes. Endosomes derived from slime mold lacking inositol pyrophosphates also display reduced dynein-directed microtubule transport. We demonstrate that Ser51 in the dynein intermediate chain (IC) is a target for pyrophosphorylation by IP7, and this modification promotes the interaction of the IC N-terminus with the p150Glued subunit of dynactin. IC–p150Glued interaction is decreased, and IC recruitment to membranes is reduced in cells lacking IP6K1. Our study provides the first evidence for the involvement of IP6Ks in dynein function and proposes that inositol pyrophosphate-mediated pyrophosphorylation may act as a regulatory signal to enhance dynein-driven transport.
Highlights
Inositol pyrophosphates are small-molecule second messengers composed of an inositol ring containing pyrophosphate groups in addition to monophosphates [1,2]
To investigate the role of inositol pyrophosphates in dynein-dependent vesicle transport, we used mouse embryonic fibroblasts (MEFs) derived from Ip6k1−/− mice, in which diphosphoinositol pentakisphosphate (IP7) levels are reduced to ∼30% compared with Ip6k1+/+ MEFs [20]
When catalytically active inositol hexakisphosphate kinase 1 (IP6K1) was expressed in Ip6k1−/− MEFs, it restored the subcellular distribution of Tfn, whereas expression of similar levels of the kinase-dead IP6K1 mutant enzyme (IP6K1K226A/S334A) [17] had no effect (Figure 1A,B)
Summary
Inositol pyrophosphates are small-molecule second messengers composed of an inositol ring containing pyrophosphate groups in addition to monophosphates [1,2]. Inositol pyrophosphates can modulate protein function in two ways: (a) by direct binding to a target protein or (b) by conferring a posttranslational modification known as pyrophosphorylation [8,9] The latter mechanism involves the transfer of a high-energy β-phosphate from an inositol pyrophosphate such as 5-IP7 to a phosphorylated serine residue to form pyrophosphoserine and has been shown to regulate protein–protein interactions [10,11]. Long-range transport in mammalian cells occurs on the microtubule cytoskeleton and is driven by two classes of motor proteins These are kinesins, which move vesicles towards the plus-end of microtubules extending to the cell periphery and cytoplasmic dynein, which carries vesicles towards the minus-end near the nucleus [13]. There have been no studies investigating the role, if any, played by inositol pyrophosphates in dynein-driven vesicular transport
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