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1.
EMBO Rep ; 24(11): e56864, 2023 11 06.
Article in English | MEDLINE | ID: mdl-37575008

ABSTRACT

Kinesin-driven intracellular transport is essential for various cell biological events and thus plays a crucial role in many pathological processes. However, little is known about the molecular basis of the specific and dynamic cargo-binding mechanism of kinesins. Here, an integrated structural analysis of the KIF3/KAP3 and KIF3/KAP3-APC complexes unveils the mechanism by which KIF3/KAP3 can dynamically grasp APC in a two-step manner, which suggests kinesin-cargo recognition dynamics composed of cargo loading, locking, and release. Our finding is the first demonstration of the two-step cargo recognition and stabilization mechanism of kinesins, which provides novel insights into the intracellular trafficking machinery.


Subject(s)
Cell Communication , Kinesins , Kinesins/metabolism , Biological Transport , Microtubules/metabolism
2.
EMBO J ; 41(5): e108899, 2022 03 01.
Article in English | MEDLINE | ID: mdl-35132656

ABSTRACT

The mechanochemical coupling of ATPase hydrolysis and conformational dynamics in kinesin motors facilitates intramolecular interaction cycles between the kinesin motor and neck domains, which are essential for microtubule-based motility. Here, we characterized a charge-inverting KIF1A-E239K mutant that we identified in a family with axonal-type Charcot-Marie-Tooth disease and also in 24 cases in human neuropathies including spastic paraplegia and hereditary sensory and autonomic neuropathy. We show that Glu239 in the ß7 strand is a key residue of the motor domain that regulates the motor-neck interaction. Expression of the KIF1A-E239K mutation has decreased ability to complement Kif1a+/- neurons, and significantly decreases ATPase activity and microtubule gliding velocity. X-ray crystallography shows that this mutation causes an excess positive charge on ß7, which may electrostatically interact with a negative charge on the neck. Quantitative mass spectrometric analysis supports that the mutation hyper-stabilizes the motor-neck interaction at the late ATP hydrolysis stage. Thus, the negative charge of Glu239 dynamically regulates the kinesin motor-neck interaction, promoting release of the neck from the motor domain upon ATP hydrolysis.


Subject(s)
Adenosine Triphosphatases/genetics , Kinesins/genetics , Mutation/genetics , Neurons/physiology , Aged , Amino Acid Sequence , Axons/physiology , Charcot-Marie-Tooth Disease , Humans , Male , Microtubules/genetics , Middle Aged , Sequence Alignment
3.
Cell Rep ; 35(2): 108971, 2021 04 13.
Article in English | MEDLINE | ID: mdl-33852848

ABSTRACT

In schizophrenia (SCZ), neurons in the brain tend to undergo gross morphological changes, but the related molecular mechanism remains largely elusive. Using Kif3b+/- mice as a model with SCZ-like behaviors, we found that a high-betaine diet can significantly alleviate schizophrenic traits related to neuronal morphogenesis and behaviors. According to a deficiency in the transport of collapsin response mediator protein 2 (CRMP2) by the KIF3 motor, we identified a significant reduction in lamellipodial dynamics in developing Kif3b+/- neurons as a cause of neurite hyperbranching. Betaine administration significantly decreases CRMP2 carbonylation, which enhances the F-actin bundling needed for proper lamellipodial dynamics and microtubule exclusion and may thus functionally compensate for KIF3 deficiency. Because the KIF3 expression levels tend to be downregulated in the human prefrontal cortex of the postmortem brains of SCZ patients, this mechanism may partly participate in human SCZ pathogenesis, which we hypothesize could be alleviated by betaine administration.


Subject(s)
Betaine/pharmacology , Intercellular Signaling Peptides and Proteins/genetics , Kinesins/genetics , Nerve Tissue Proteins/genetics , Neurons/drug effects , Prefrontal Cortex/drug effects , Pseudopodia/drug effects , Schizophrenia/diet therapy , Actins/genetics , Actins/metabolism , Animals , Behavior, Animal/drug effects , Biological Transport , Diet/methods , Disease Models, Animal , Gene Expression Regulation, Developmental , Humans , Intercellular Signaling Peptides and Proteins/deficiency , Kinesins/deficiency , Male , Mice , Mice, Knockout , Microtubules/drug effects , Microtubules/metabolism , Microtubules/ultrastructure , Nerve Tissue Proteins/deficiency , Neurons/metabolism , Neurons/ultrastructure , Prefrontal Cortex/metabolism , Prefrontal Cortex/pathology , Protein Binding , Protein Carbonylation , Pseudopodia/metabolism , Pseudopodia/ultrastructure , Schizophrenia/genetics , Schizophrenia/metabolism , Schizophrenia/pathology
4.
Life Sci Alliance ; 2(5)2019 10.
Article in English | MEDLINE | ID: mdl-31591136

ABSTRACT

Enhanced carbonyl stress underlies a subset of schizophrenia, but its causal effects remain elusive. Here, we elucidated the molecular mechanism underlying the effects of carbonyl stress in iPS cells in which the gene encoding zinc metalloenzyme glyoxalase I (GLO1), a crucial enzyme for the clearance of carbonyl stress, was disrupted. The iPS cells exhibited significant cellular and developmental deficits, and hyper-carbonylation of collapsing response mediator protein 2 (CRMP2). Structural and biochemical analyses revealed an array of multiple carbonylation sites in the functional motifs of CRMP2, particularly D-hook (for dimerization) and T-site (for tetramerization), which are critical for the activity of the CRMP2 tetramer. Interestingly, carbonylated CRMP2 was stacked in the multimer conformation by irreversible cross-linking, resulting in loss of its unique function to bundle microtubules. Thus, the present study revealed that the enhanced carbonyl stress stemmed from the genetic aberrations results in neurodevelopmental deficits through the formation of irreversible dysfunctional multimer of carbonylated CRMP2.


Subject(s)
Frameshift Mutation , Intercellular Signaling Peptides and Proteins/chemistry , Intercellular Signaling Peptides and Proteins/metabolism , Lactoylglutathione Lyase/genetics , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/metabolism , Schizophrenia/genetics , Cell Differentiation , Cells, Cultured , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Mass Spectrometry , Models, Molecular , Protein Carbonylation , Protein Conformation , Protein Multimerization , Schizophrenia/metabolism
5.
Cell Rep ; 28(9): 2413-2426.e7, 2019 08 27.
Article in English | MEDLINE | ID: mdl-31461655

ABSTRACT

The axon initial segment (AIS) is a compartment that serves as a molecular barrier to achieve axon-dendrite differentiation. Distribution of specific proteins during early neuronal development has been proposed to be critical for AIS construction. However, it remains unknown how these proteins are specifically targeted to the proximal axon within this limited time period. Here, we reveal spatiotemporal regulation driven by the microtubule (MT)-based motor KIF3A/B/KAP3 that transports TRIM46, influenced by a specific MARK2 phosphorylation cascade. In the proximal part of the future axon under low MARK2 activity, the KIF3/KAP3 motor recognizes TRIM46 as cargo and transports it to the future AIS. In contrast, in the somatodendritic area under high MARK2 activity, KAP3 phosphorylated at serine 60 by MARK2 cannot bind with TRIM46 and be transported. This spatiotemporal regulation between KIF3/KAP3 and TRIM46 under specific MARK2 activity underlies the specific transport needed for axonal differentiation.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Axonal Transport , Axons/metabolism , Cytoskeletal Proteins/metabolism , Kinesins/metabolism , Nerve Tissue Proteins/metabolism , Neurogenesis , Animals , COS Cells , Chlorocebus aethiops , Female , HEK293 Cells , Humans , MAP Kinase Signaling System , Male , Mice , Mice, Inbred C57BL , Mice, Inbred ICR , Mitogen-Activated Protein Kinase 1/metabolism
6.
Biophys Rev ; 10(2): 299-306, 2018 Apr.
Article in English | MEDLINE | ID: mdl-29204883

ABSTRACT

The need for accurate description of protein behavior in solution has gained importance in various fields, including biophysics, biochemistry, structural biology, drug discovery, and antibody drugs. To achieve the desired accuracy, multiple precise analyses should be performed on the target molecule, compared, and effectively combined. This review focuses on the combination of multiple analyses in solution: size-exclusion chromatography (SEC), multi-angle light scattering (MALS), small-angle X-ray scattering (SAXS), analytical ultracentrifugation (AUC), and their complementary methods, such as atomic force microscopy (AFM) and mass spectrometry (MS). We also discuss the comparison between the determined molar mass value of not only the standard proteins, but of a target molecule tubulin and its depolymerizing protein, KIF2, as an example. The comparison of the estimated molar mass value from the different methods provides additional information about the target molecule, because the value reflects the dynamically changing states of the target molecule in solution. The combination and integration of multiple methods will permit a deeper understanding of protein dynamics in solution.

7.
Cell Rep ; 20(11): 2626-2638, 2017 Sep 12.
Article in English | MEDLINE | ID: mdl-28903043

ABSTRACT

Microtubules (MTs) are dynamic structures that are fundamental for cell morphogenesis and motility. MT-associated motors work efficiently to perform their functions. Unlike other motile kinesins, KIF2 catalytically depolymerizes MTs from the peeled protofilament end during ATP hydrolysis. However, the detailed mechanism by which KIF2 drives processive MT depolymerization remains unknown. To elucidate the catalytic mechanism, the transitional KIF2-tubulin complex during MT depolymerization was analyzed through multiple methods, including atomic force microscopy, size-exclusion chromatography, multi-angle light scattering, small-angle X-ray scattering, analytical ultracentrifugation, and mass spectrometry. The analyses outlined the conformation in which one KIF2core domain binds tightly to two tubulin dimers in the middle pre-hydrolysis state during ATP hydrolysis, a process critical for catalytic MT depolymerization. The X-ray crystallographic structure of the KIF2core domain displays the activated conformation that sustains the large KIF2-tubulin 1:2 complex.


Subject(s)
Biocatalysis , Kinesins/chemistry , Kinesins/metabolism , Microtubules/metabolism , Polymerization , Tubulin/chemistry , Tubulin/metabolism , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Chromatography, Gel , Hydrolysis , Kinesins/genetics , Loss of Function Mutation , Models, Molecular , Molecular Weight , Protein Binding , Protein Conformation , Protein Multimerization , Scattering, Small Angle , X-Ray Diffraction
8.
Cell Rep ; 12(11): 1774-88, 2015 Sep 22.
Article in English | MEDLINE | ID: mdl-26344760

ABSTRACT

Neurons exhibit dynamic structural changes in response to extracellular stimuli. Microtubules (MTs) provide rapid and dramatic cytoskeletal changes within the structural framework. However, the molecular mechanisms and signaling networks underlying MT dynamics remain unknown. Here, we have applied a comprehensive and quantitative phospho-analysis of the MT destabilizer KIF2A to elucidate the regulatory mechanisms of MT dynamics within neurons in response to extracellular signals. Interestingly, we identified two different sets of KIF2A phosphorylation profiles that accelerate (A-type) and brake (B-type) the MT depolymerization activity of KIF2A. Brain-derived neurotrophic factor (BDNF) stimulates PAK1 and CDK5 kinases, which decrease the MT depolymerizing activity of KIF2A through B-type phosphorylation, resulting in enhanced outgrowth of neural processes. In contrast, lysophosphatidic acid (LPA) induces ROCK2 kinase, which suppresses neurite outgrowth from round cells via A-type phosphorylation. We propose that these two mutually exclusive forms of KIF2A phosphorylation differentially regulate neuronal morphogenesis during development.


Subject(s)
Kinesins/metabolism , Microtubules/metabolism , Neurons/cytology , Neurons/metabolism , Repressor Proteins/metabolism , Amino Acid Sequence , Animals , COS Cells , Cell Differentiation/physiology , Chlorocebus aethiops , Mice , Molecular Sequence Data , Morphogenesis , Phosphorylation
9.
Neuron ; 87(5): 1022-35, 2015 Sep 02.
Article in English | MEDLINE | ID: mdl-26335646

ABSTRACT

A regulated mechanism of cargo loading is crucial for intracellular transport. N-cadherin, a synaptic adhesion molecule that is critical for neuronal function, must be precisely transported to dendritic spines in response to synaptic activity and plasticity. However, the mechanism of activity-dependent cargo loading remains unclear. To elucidate this mechanism, we investigated the activity-dependent transport of N-cadherin via its transporter, KIF3A. First, by comparing KIF3A-bound cargo vesicles with unbound KIF3A, we identified critical KIF3A phosphorylation sites and specific kinases, PKA and CaMKIIa, using quantitative phosphoanalyses. Next, mutagenesis and kinase inhibitor experiments revealed that N-cadherin transport was enhanced via phosphorylation of the KIF3A C terminus, thereby increasing cargo-loading activity. Furthermore, N-cadherin transport was enhanced during homeostatic upregulation of synaptic strength, triggered by chronic inactivation by TTX. We propose the first model of activity-dependent cargo loading, in which phosphorylation of the KIF3A C terminus upregulates the loading and transport of N-cadherin in homeostatic synaptic plasticity.


Subject(s)
Brain/cytology , Cadherins/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Gene Expression Regulation, Enzymologic/physiology , Kinesins/metabolism , Neurons/metabolism , Adenosine Triphosphate/pharmacology , Anesthetics, Local/pharmacology , Animals , Calcium/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Type 2/genetics , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Cyclic AMP-Dependent Protein Kinases/genetics , Enzyme Inhibitors/pharmacology , Gene Expression Regulation/drug effects , Gene Expression Regulation/physiology , Gene Expression Regulation, Enzymologic/genetics , Kinesins/genetics , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Mice , Mice, Transgenic , Peptide Fragments/metabolism , Phosphorylation/genetics , Protein Transport/drug effects , Protein Transport/physiology , Synapses/drug effects , Synapses/metabolism , Tetrodotoxin/pharmacology , Time Factors
10.
Cell ; 116(4): 591-602, 2004 Feb 20.
Article in English | MEDLINE | ID: mdl-14980225

ABSTRACT

Unlike other kinesins, middle motor domain-type kinesins depolymerize the microtubule from its ends. To elucidate its mechanism, we solved the X-ray crystallographic structure of KIF2C, a murine member of this family. Three major class-specific features were identified. The class-specific N-terminal neck adopts a long and rigid helical structure extending out vertically into the interprotofilament groove. This structure explains its dual roles in targeting to the end of the microtubule and in destabilization of the lateral interaction of the protofilament. The loop L2 forms a unique finger-like structure, long and rigid enough to reach the next tubulin subunit to stabilize the peeling of the protofilament. The open conformation of the switch I loop could be reversed by the shift of the microtubule binding L8 loop, suggesting its role as the sensor to trigger ATP hydrolysis. Mutational analysis supports these structural implications.


Subject(s)
Kinesins/chemistry , Microtubules/chemistry , Adenosine Triphosphate/chemistry , Amino Acid Sequence , Animals , Binding Sites , COS Cells , Catalysis , Crystallography, X-Ray , DNA Mutational Analysis , Microtubules/metabolism , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Conformation , Protein Structure, Secondary , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Transfection
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