p150Glued Subunit Of Dynactin Research Articles

Selective expression of neurodegenerative diseases-related mutant p150Glued in midbrain dopaminergic neurons causes progressive degeneration of nigrostriatal pathway

Aim: Missense mutations of dynactin subunit p150Glued have been associated with multiple neurodegenerative diseases, including Perry syndrome, characterized by inherited parkinsonism, depression, weight loss, and hypoventilation. The current study investigated how the pathogenic mutant p150Glued affects the integrity and function of the nigrostriatal dopaminergic (DA) pathway in vivo. Methods: Using a tetracycline-controlled transcriptional regulation system, transgenic mouse models were generated with selective overexpression of wild-type, motor neuron disease-related G59S mutant, or Perry syndrome-related G71R mutant human p150Glued in midbrain DA neurons. A series of behavioral, neuropathological, neurochemical, electrochemical, and biochemical studies were performed on the mice to examine and compare the pathogenic impact of the two mutant p150Glued on the survival and function of midbrain DA neurons. Results: Compared with non-transgenic control mice, transgenic mice overexpressing wild-type human p150Glued showed neither motor phenotypes nor pathological, functional, or biochemical abnormalities of the nigrostriatal DA pathway. Transgenic mice overexpressing G59S mutant p150Glued displayed weight loss, motor deficits, early-onset defects in dopamine transmission, and early-onset loss of DA neurons and axons. Transgenic mice overexpressing G71R p150Glued mutant exhibited hyperactivities, impaired motor coordination, early-onset dysfunction of dopamine uptake, and late-onset loss of DA neurons and axons. In addition, overexpression of either G59S or G71R mutant p150Glued in midbrain DA neurons preferentially downregulated the expression of dopamine transporter at dopaminergic axon terminals. Furthermore, G59S mutant p150Glued rather than G71R mutant p150Glued formed aggregates in midbrain DA neurons in vivo, and the aggregates trapped dynein/dynactin, co-localized with lysosomes, and upregulated ubiquitination. Conclusion: These findings demonstrate that selective expression of either G59S or G71R mutant p150Glued in mouse midbrain DA neurons leads to progressive degeneration of the nigrostriatal DA pathway and indicate that G59S and G71R mutant p150Glued exhibit differential pathogenic impact on the survival and function of midbrain DA neurons in vivo.

Interplay of Disorder and Sequence Specificity in the Formation of Stable Dynein-Dynactin Complexes

Cytoplasmic dynein is a eukaryotic motor protein complex that, along with its regulatory protein dynactin, is essential to the transport of organelles within cells. The interaction of dynein with dynactin is regulated by binding between the intermediate chain (IC) subunit of dynein and the p150Glued subunit of dynactin. Even though in the rat versions of these proteins this interaction primarily involves the single α-helix region at the N-terminus of the IC, in Drosophila and yeast ICs the removal of a nascent helix (H2) downstream of the single α-helix considerably diminishes IC-p150Glued complex stability. We find that for ICs from various species, there is a correlation between disorder in H2 and its contribution to binding affinity, and that sequence variations in H2 that do not change the level of disorder show similar binding behavior. Analysis of the structure and interactions of the IC from Chaetomium thermophilum demonstrates that the H2 region of C. thermophilum IC has a low helical propensity and establishes that H2 binds directly to the coiled-coil 1B (CC1B) domain of p150Glued, thus explaining why H2 is necessary for tight binding. Isothermal titration calorimetry, circular dichroism, and NMR studies of smaller CC1B constructs localize the region of CC1B most essential for a tight interaction with IC. These results suggest that it is the level of disorder in H2 of IC along with its charge, rather than sequence specificity, that underlie its importance in initiating tight IC-p150Glued complex formation. We speculate that the nascent H2 helix may provide conformational flexibility to initiate binding, whereas those species that have a fully folded H2 have co-opted an alternative mechanism for promoting p150Glued binding.

Retrograde Degenerative Signaling Mediated by the p75 Neurotrophin Receptor Requires p150Glued Deacetylation by Axonal HDAC1

During development, neurons undergo apoptosis if they do not receive adequate trophic support from tissues they innervate or when detrimental factors activate the p75 neurotrophin receptor (p75NTR) at their axon ends. Trophic factor deprivation (TFD) or activation of p75NTR in distal axons results in a retrograde degenerative signal. However, the nature of this signal and the regulation of its transport are poorly understood. Here, we identify p75NTR intracellular domain (ICD) and histone deacetylase 1 (HDAC1) as part of a retrograde pro-apoptotic signal generated in response to TFD or ligand binding to p75NTR in sympathetic neurons. We report an unconventional function of HDAC1 in retrograde transport of a degenerative signal and its constitutive presence in sympathetic axons. HDAC1 deacetylates dynactin subunit p150Glued, which enhances its interaction with dynein. These findings define p75NTR ICD as a retrograde degenerative signal and reveal p150Glued deacetylation as a unique mechanism regulating axonal transport.

MISP regulates the IQGAP1/Cdc42 complex to collectively orchestrate spindle orientation and mitotic progression

Precise mitotic spindle orientation is essential for both cell fate and tissue organization while defects in this process are associated with tumorigenesis and other diseases. In most animal cell types, the dynein motor complex is anchored at the cell cortex and exerts pulling forces on astral microtubules to position the spindle. The actin-binding protein MISP controls spindle orientation and mitotic progression in human cells. However, the exact underlying mechanism remains to be elucidated. Here we report that MISP interacts with the multidomain scaffolding protein IQGAP1. We further show that MISP binds to the active form of Cdc42 through IQGAP1. Depletion of MISP promotes increased accumulation of IQGAP1 at the cell cortex and a decrease in its Cdc42-binding capacity leading to reduced active Cdc42 levels. Interestingly, overexpression of IQGAP1 can rescue mitotic defects caused by MISP downregulation including spindle misorientation, loss of astral microtubules and prolonged mitosis and also restores active Cdc42 levels. Importantly, we find that IQGAP1 acts downsteam of MISP in regulating astral microtubule dynamics and the localization of the dynactin subunit p150glued that is crucial for proper spindle positioning. We propose that MISP regulates IQGAP1 and Cdc42 to ensure proper mitotic progression and correct spindle orientation.

Inositol hexakisphosphate kinase 1 (IP6K1) activity is required for cytoplasmic dynein-driven transport.

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.

Dominant-Negative Effects of Adult-Onset Huntingtin Mutations Alter the Division of Human Embryonic Stem Cells-Derived Neural Cells.

Mutations of the huntingtin protein (HTT) gene underlie both adult-onset and juvenile forms of Huntington’s disease (HD). HTT modulates mitotic spindle orientation and cell fate in mouse cortical progenitors from the ventricular zone. Using human embryonic stem cells (hESC) characterized as carrying mutations associated with adult-onset disease during pre-implantation genetic diagnosis, we investigated the influence of human HTT and of an adult-onset HD mutation on mitotic spindle orientation in human neural stem cells (NSCs) derived from hESCs. The RNAi-mediated silencing of both HTT alleles in neural stem cells derived from hESCs disrupted spindle orientation and led to the mislocalization of dynein, the p150Glued subunit of dynactin and the large nuclear mitotic apparatus (NuMA) protein. We also investigated the effect of the adult-onset HD mutation on the role of HTT during spindle orientation in NSCs derived from HD-hESCs. By combining SNP-targeting allele-specific silencing and gain-of-function approaches, we showed that a 46-glutamine expansion in human HTT was sufficient for a dominant-negative effect on spindle orientation and changes in the distribution within the spindle pole and the cell cortex of dynein, p150Glued and NuMA in neural cells. Thus, neural derivatives of disease-specific human pluripotent stem cells constitute a relevant biological resource for exploring the impact of adult-onset HD mutations of the HTT gene on the division of neural progenitors, with potential applications in HD drug discovery targeting HTT-dynein-p150Glued complex interactions.

Cytoplasmic dynein and its regulatory proteins in Golgi pathology in nervous system disorders.

The Golgi apparatus is a dynamic organelle involved in processing and sorting of lipids and proteins. In neurons, the Golgi apparatus is important for the development of axons and dendrites and maintenance of their highly complex polarized morphology. The motor protein complex cytoplasmic dynein has an important role in Golgi apparatus positioning and function. Together, with dynactin and other regulatory factors it drives microtubule minus-end directed motility of Golgi membranes. Inhibition of dynein results in fragmentation and dispersion of the Golgi ribbon in the neuronal cell body, resembling the Golgi abnormalities observed in some neurodegenerative disorders, in particular motor neuron diseases. Mutations in dynein and its regulatory factors, including the dynactin subunit p150Glued, BICD2 and Lis-1, are associated with several human nervous system disorders, including cortical malformation and motor neuropathy. Here we review the role of dynein and its regulatory factors in Golgi function and positioning, and the potential role of dynein malfunction in causing Golgi apparatus abnormalities in nervous system disorders.

Protein Disorder in Dynein Regulation by Dynactin and NudE

Cytoplasmic dynein is a multi-subunit protein complex responsible for retrograde transport of diverse cellular cargoes along microtubules. Dynein is comprised of heavy chains responsible for motor activity, and intermediate chain (IC) and light chains for cargo attachment and regulation. Dynein light chain LC8 is conserved across species and its binding promotes dimerization and stabilization of IC but its effect on dynein regulation remains unclear. Dynein activity is regulated by other proteins such as dynactin, which is essential for most dynein activities; and NudE, which functions in targeting dynein to the kinetochores. LC8, dynactin subunit p150Glued and NudE coiled-coil domain all bind the disordered N-terminal domain of IC. Using NMR and ITC on Saccharomyces cerevisiae and Drosophila melanogaster constructs, we show that the N-terminal helix of IC is a single α-helix (SAH) instead of a dimeric coiled-coil as predicted. NMR titrations map the exact residues of IC involved in binding to both p150Glued and NudE and the accompanying changes in structure and dynamics, while ITC experiments identify the domains of IC necessary for full binding affinity. In yeast, p150 and NudE both bind the IC SAH domain, and both interactions are enhanced when LC8 is present. In Drosophila, p150 and NudE both bind to the SAH domain but p150 also binds to a second site on IC, a nascent helix separated from the SAH domain by a 4-residue linker. A dimeric IC formed by cross-linking IC chains has increased affinity to p150 but similar affinity to NudE, indicating that bivalency causes differential effects on binding regulatory proteins, and illustrating intriguing species variation in dynactin binding to IC. These studies illustrate the importance of multiple techniques to elucidate interplay of order and disorder in providing both structural and functional versatility for complicated systems.

Dynactin Functions as Both a Dynamic Tether and Brake during Dynein-Driven Motility

Dynactin is a large multi-subunit complex that is an important cofactor for cytoplasmic dynein in cellular processes including organelle trafficking and mitosis. Dynactin increases the processivity of dynein and also targets dynein to specific cellular locations. Although the role of dynactin in dynein-mediated processes is well established, the underlying mechanisms involved are unknown. Here, we use single molecule approaches to investigate the contribution of the dynactin subunit p150Glued to dynein motor function. We first characterized the motility of purified GFP-tagged mammalian dynein isolated from a knock-in mouse brain tissue (Zhang et al., 2013). Consistent with previous reports (Mallik et al., 2005; Ross et al., 2006), single molecules of dynein-GFP switch stochastically from processive to diffusive states of motion. We analyzed this motion by a novel algorithm that we developed to parse trajectories into different states and found that dynein is processive 60% of the time. To examine the activation of dynein by dynactin, we investigated the formation and co-migration of a dynein-p150Glued co-complex using dual-color TIRF microscopy. We provide direct evidence that p150Glued is sufficient to recruit and tether dynein to the microtubule and that this recruitment is concentration-dependent; p150Glued increases the on-rate and decreases the off-rate of dynein from microtubules. Single molecule imaging of motility in cell extracts demonstrates that the CAP-Gly domain of dynactin is essential for the decreased detachment rate of the dynein-dynactin complex from the microtubule and also acts as a brake to slow the dynein motor. Consistent with this important role, two neurodegenerative disease-causing mutations in the CAP-Gly domain abrogate these functions in our assays. Together, these observations support a model in which dynactin enhances the initial recruitment of dynein onto microtubules and promotes the sustained engagement of dynein with its cytoskeletal track.

Reconstitution of a hierarchical +TIP interaction network controlling microtubule end tracking of dynein.

Growing microtubule end regions recruit a variety of proteins collectively termed +TIPs, which confer local functions to the microtubule cytoskeleton. +TIPs form dynamic interaction networks whose behaviour depends on a number of potentially competitive and hierarchical interaction modes. The rules that determine which of the various +TIPs are recruited to the limited number of available binding sites at microtubule ends remain poorly understood. Here we examined how the human dynein complex, the main minus-end-directed motor and an important +TIP (refs, , ), is targeted to growing microtubule ends in the presence of different +TIP competitors. Using a total internal reflection fluorescence microscopy-based reconstitution assay, we found that a hierarchical recruitment mode targets the large dynactin subunit p150Glued to growing microtubule ends via EB1 and CLIP-170 in the presence of competing SxIP-motif-containing peptides. We further show that the human dynein complex is targeted to growing microtubule ends through an interaction of the tail domain of dynein with p150Glued. Our results highlight how the connectivity and hierarchy within dynamic +TIP networks areorchestrated.

Mutations in cytoplasmic dynein and its regulators cause malformations of cortical development and neurodegenerative diseases

Neurons are highly specialized for the processing and transmission of electrical signals and use cytoskeleton-based motor proteins to transport different vesicles and cellular materials. Abnormalities in intracellular transport are thought to be a critical factor in the degeneration and death of neurons in both the central and peripheral nervous systems. Several recent studies describe disruptive mutations in the minus-end-directed microtubule motor cytoplasmic dynein that are directly linked to human motor neuropathies, such as SMA (spinal muscular atrophy) and axonal CMT (Charcot-Marie-Tooth) disease or malformations of cortical development, including lissencephaly, pachygyria and polymicrogyria. In addition, genetic defects associated with these and other neurological disorders have been found in multifunctional adaptors that regulate dynein function, including the dynactin subunit p150(Glued), BICD2 (Bicaudal D2), Lis-1 (lissencephaly 1) and NDE1 (nuclear distribution protein E). In the present paper we provide an overview of the disease-causing mutations in dynein motors and regulatory proteins that lead to a broad phenotypic spectrum extending from peripheral neuropathies to cerebral malformations.

JIP1 regulates the directionality of APP axonal transport by coordinating kinesin and dynein motors

Regulation of the opposing kinesin and dynein motors that drive axonal transport is essential to maintain neuronal homeostasis. Here, we examine coordination of motor activity by the scaffolding protein JNK-interacting protein 1 (JIP1), which we find is required for long-range anterograde and retrograde amyloid precursor protein (APP) motility in axons. We identify novel interactions between JIP1 and kinesin heavy chain (KHC) that relieve KHC autoinhibition, activating motor function in single molecule assays. The direct binding of the dynactin subunit p150(Glued) to JIP1 competitively inhibits KHC activation in vitro and disrupts the transport of APP in neurons. Together, these experiments support a model whereby JIP1 coordinates APP transport by switching between anterograde and retrograde motile complexes. We find that mutations in the JNK-dependent phosphorylation site S421 in JIP1 alter both KHC activation in vitro and the directionality of APP transport in neurons. Thus phosphorylation of S421 of JIP1 serves as a molecular switch to regulate the direction of APP transport in neurons.

Kif3a interacts with Dynactin subunit p150Glued to organize centriole subdistal appendages

Formation of cilia, microtubule-based structures that function in propulsion and sensation, requires Kif3a, a subunit of Kinesin II essential for intraflagellar transport (IFT). We have found that, Kif3a is also required to organize centrioles. In the absence of Kif3a, the subdistal appendages of centrioles are disorganized and lack p150(Glued) and Ninein. Consequently, microtubule anchoring, centriole cohesion and basal foot formation are abrogated by loss of Kif3a. Kif3a localizes to the mother centriole and interacts with the Dynactin subunit p150(Glued). Depletion of p150(Glued) phenocopies the effects of loss of Kif3a, indicating that Kif3a recruitment of p150(Glued) is critical for subdistal appendage formation. The transport functions of Kif3a are dispensable for subdistal appendage organization as mutant forms of Kif3a lacking motor activity or the motor domain can restore p150(Glued) localization. Comparison to cells lacking Ift88 reveals that the centriolar functions of Kif3a are independent of IFT. Thus, in addition to its ciliogenic roles, Kif3a recruits p150(Glued) to the subdistal appendages of mother centrioles, critical for centrosomes to function as microtubule-organizing centres.

JIP1 Sustains Long Distance Anterograde Transport via Direct Regulation of Kinesin Autoinhibition and Coordination of Retrograde Motor Association

Analysis of the highly processive vesicular cargo APP (amyloid precursor protein) in primary mammalian neurons shows axonal transport characterized by long run lengths with few pauses or directional switches. In order to elucidate the molecular mechanism for sustaining long-distance processive motility, we focused on the adaptor protein JIP1, which binds directly to APP and KLC (kinesin light chain). Upon siRNA depletion of JIP1 from primary neurons, we observed significant defects in both anterograde and retrograde APP transport. We further investigated the association of JIP1 with kinesin-1 and identified novel and distinct interactions between JIP1 and both kinesin heavy chain (KHC) stalk and tail. Using in vitro TIRF motility assays with single-molecule resolution, we found that addition of JIP1 relieves autoinhibition of KHC, leading to activation of motility and increased run frequency, run length and speed. Interestingly, truncated constructs of JIP1 that only bind KHC stalk or KHC tail were sufficient to initiate processive runs, but could not fully recapitulate the enhancement of KHC run length or speed observed with full-length JIP1. In addition, JIP1 can coordinate both kinesin- and dynein-driven motility, as we identify a novel interaction between JIP1 and the p150Glued subunit of dynactin. Both in vitro and in primary neurons, addition of the JIP1-binding region of p150Glued competitively inhibits JIP1-mediated enhancement of KHC processivity. In contrast to a stochastic tug-of-war model, our data suggest that JIP1 sustains long-distance transport by coordinating the formation of two distinct motile complexes - an anterograde complex that binds directly to KHC and a retrograde complex via its interaction with dynactin.

The dynactin p150 subunit: cell biology studies of sequence changes found in ALS/MND and Parkinsonian Syndromes

The dynactin p150glued subunit, encoded by the gene DCTN1 is part of the dynein-dynactin motor protein complex responsible for retrograde axonal transport. This subunit is a candidate modifier for neurodegenerative diseases, in particular motoneuron and extrapyramidal diseases. Based on an extensive screening effort of all 32 exons in more than 2,500 ALS/MND patients, patients suffering from Parkinsonian Syndromes and controls, we investigated 24 sequence variants of p150 in cell-based studies. We used both non-neuronal cell lines and primary rodent spinal motoneurons and report on cell biological abnormalities in five of these sequence alterations and also briefly report on the clinical features. Our results suggest the presence of biological changes caused by some p150 mutants pointing to a potential pathogenetic significance as modifier of the phenotype of the human disease.

Tubulin-binding cofactor B is a direct interaction partner of the dynactin subunit p150Glued

The dynactin p150(Glued) subunit, encoded by the gene DCTN1, is part of the dynein-dynactin motor protein complex responsible for retrograde axonal transport in motor neurons. The p150 subunit is a candidate gene for neurodegenerative diseases, in particular motor neuron and extrapyramidal diseases. Tubulin-binding cofactors are believed to be involved in tubulin biogenesis and degradation and therefore to contribute to microtubule functional diversity and regulation. A yeast-two-hybrid screen for putative interacting proteins of dynactin p150(Glued) has revealed tubulin-folding cofactor B (TBCB). We analyzed the interaction of these proteins and investigated the impact of this complex on the microtubule network in cell lines and primary hippocampal neurons in vitro. We especially concentrated on neuronal morphology and synaptogenesis. Overexpression of both proteins or depletion of TBCB alone does not alter the microtubule network and/or neuronal morphology. The demonstration of the interaction of the transport molecule dynactin and the tubulin-regulating factor TBCB is thought to have an impact on several cellular mechanisms. TBCB expression levels have been found to have only a subtle influence on the microtubule network and neuronal morphology. However, overexpression of TBCB leads to the decreased localization of p150 to the microtubule network that might result in a functional modulation of this protein complex.

Neurodegenerative Mutation in Cytoplasmic Dynein Alters Its Organization and Dynein-Dynactin and Dynein-Kinesin Interactions

A single amino acid change, F580Y (Legs at odd angles (Loa), Dync1h1Loa), in the highly conserved and overlapping homodimerization, intermediate chain, and light intermediate chain binding domain of the cytoplasmic dynein heavy chain can cause severe motor and sensory neuron loss in mice. The mechanism by which the Loa mutation impairs the neuron-specific functions of dynein is not understood. To elucidate the underlying molecular mechanisms of neurodegeneration arising from this mutation, we applied a cohort of biochemical methods combined with in vivo assays to systemically study the effects of the mutation on the assembly of dynein and its interaction with dynactin. We found that the Loa mutation in the heavy chain leads to increased affinity of this subunit of cytoplasmic dynein to light intermediate and a population of intermediate chains and a suppressed association of dynactin to dynein. These data suggest that the Loa mutation drives the assembly of cytoplasmic dynein toward a complex with lower affinity to dynactin and thus impairing transport of cargos that tether to the complex via dynactin. In addition, we detected up-regulation of kinesin light chain 1 (KLC1) and its increased association with dynein but reduced microtubule-associated KLC1 in the Loa samples. We provide a model describing how up-regulation of KLC1 and its interaction with cytoplasmic dynein in Loa could play a regulatory role in restoring the retrograde and anterograde transport in the Loa neurons.

Par6α Interacts with the Dynactin Subunit p150Gluedand Is a Critical Regulator of Centrosomal Protein Recruitment

The centrosome contains proteins that control the organization of the microtubule cytoskeleton in interphase and mitosis. Its protein composition is tightly regulated through selective and cell cycle-dependent recruitment, retention, and removal of components. However, the mechanisms underlying protein delivery to the centrosome are not completely understood. We describe a novel function for the polarity protein Par6α in protein transport to the centrosome. We detected Par6α at the centrosome and centriolar satellites where it interacted with the centriolar satellite protein PCM-1 and the dynactin subunit p150(Glued). Depletion of Par6α caused the mislocalization of p150(Glued) and centrosomal components that are critical for microtubule anchoring at the centrosome. As a consequence, there were severe alterations in the organization of the microtubule cytoskeleton in the absence of Par6α and cell division was blocked. We propose a model in which Par6α controls centrosome organization through its association with the dynactin subunit p150(Glued).

The Crystal Structure of Dynein Intermediate Chain-Light Chain Roadblock Complex Gives New Insights into Dynein Assembly

The roadblock/LC7 dynein light chain is a ubiquitous component of all dyneins and is essential for many diverse processes including proper axonal transport and dendrite growth. In addition, LC7 functions in non-dynein transcriptional activation of the transforming growth factor-beta complex. Crystal structures of Drosophila melanogaster LC7 in the apo form and in complex with a segment of the disordered N-terminal domain of dynein intermediate chain (IC) provide the first definitive identification of the IC sequence recognized by LC7. The site, confirmed by isothermal titration calorimetry studies, overlaps the IC sequence considered in the literature to be an IC self-association domain. The IC peptide binds as two amphipathic helices that lie along an extensive hydrophobic cleft on LC7 and ends with a polar side-chain interaction network that includes conserved residues from both proteins. The LC7 recognition sequence on IC and its interface with LC7 are well conserved and are, thus, likely representative of all IC x LC7 structures. Interestingly, the position of bound IC in the IC x LC7 complex mimics a helix that is integrated into the primary structure in distantly related LC7 homologs. The IC x LC7 structure further shows that the naturally occurring robl(Z) deletion mutation contains the majority of the IC binding site and suggests that promotion of IC binding by phosphorylation of LC7 is an indirect effect.

Phosphorylation Controls Autoinhibition of Cytoplasmic Linker Protein-170

Cytoplasmic linker protein (CLIP)-170 is a microtubule (MT) plus-end-tracking protein that regulates MT dynamics and links MT plus ends to different intracellular structures. We have shown previously that intramolecular association between the N and C termini results in autoinhibition of CLIP-170, thus altering its binding to MTs and the dynactin subunit p150(Glued) (J. Cell Biol. 2004: 166, 1003-1014). In this study, we demonstrate that conformational changes in CLIP-170 are regulated by phosphorylation that enhances the affinity between the N- and C-terminal domains. By using site-directed mutagenesis and phosphoproteomic analysis, we mapped the phosphorylation sites in the third serine-rich region of CLIP-170. A phosphorylation-deficient mutant of CLIP-170 displays an "open" conformation and a higher binding affinity for growing MT ends and p150(Glued) as compared with nonmutated protein, whereas a phosphomimetic mutant confined to the "folded back" conformation shows decreased MT association and does not interact with p150(Glued). We conclude that phosphorylation regulates CLIP-170 conformational changes resulting in its autoinhibition.