The M = 8,000 and 11,000 Outer Arm Dynein Light Chains from Chlamydomonas Flagella Have Cytoplasmic Homologues

King, Stephen M.; Patel‐King, Ramila S.

doi:10.1074/jbc.270.19.11445

Cited by 183 publications

(184 citation statements)

References 33 publications

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“…Ca 2ϩ /calmodulin activates CaM kinase II by increasing affinity for ATP as well as peptide substrates (20,24,43), suggesting that the auto-inhibitory domain suppresses binding of both substrates in the basal state. A peptide inhibitor containing residues 281-309 was shown to be competitive with respect to ATP (25) and to protect the ATP binding site from chemical modification by phenylglyoxal (44). Such an ATP-competitive mechanism is not observed in CaM kinase I (45).…”

Section: Discussionmentioning

confidence: 99%

Structural Examination of Autoregulation of Multifunctional Calcium/Calmodulin-dependent Protein Kinase II

Yang

Schulman

1999

Journal of Biological Chemistry

140

152

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Regulation of Ca2؉ /calmodulin-dependent protein kinase II is likely based on an auto-inhibitory mechanism in which a segment of the kinase occupies the catalytic site in the absence of calmodulin. We analyze potential auto-inhibitory associations by employing charge reversal and hydrophobic-to-charged residue mutagenesis. We identify interacting amino acid pairs by using double mutants to test which modification in the catalytic domain complements a given change in the auto-inhibitory domain. Our studies identify the core pseudosubstrate sequence (residues 297-300) but reveal that distinct sequences centered about the autophosphorylation site at Thr-286 are involved in the critical auto-inhibitory interactions. Individual changes in any of the residues Arg-274, His-282, Arg-283, Lys-291, Arg-297, Phe-293, and Asn-294 in the auto-inhibitory domain or their interacting partners in the catalytic domain produces an enhanced affinity for calmodulin or generates a constitutively active enzyme. A structural model of Ca 2؉ /calmodulin-dependent protein kinase II that incorporates these interactions shows that Thr-286 is oriented inwardly into a hydrophobic channel. The model explains why calmodulin must bind to the auto-inhibitory domain in order for Thr-286 in that domain to be phosphorylated and why introduction of phospho-Thr-286 produces the important Ca 2؉ -independent state of the enzyme.Multifunctional Ca 2ϩ /calmodulin-dependent protein kinase (CaM kinase) 1 II has received considerable attention because of its autoregulatory properties (reviewed in Refs. 1 and 2). Regulation of the kinase is likely based on an auto-inhibitory or pseudosubstrate mechanism in which a segment of the kinase occupies the catalytic site in the basal state. Ca 2ϩ /calmodulin activates the kinase by wrapping around its target sequence on the kinase, a site that overlaps the auto-inhibitory domain (3). The active enzyme not only phosphorylates exogenous substrates but also exhibits a prominent autophosphorylation of Thr-286 within the auto-inhibitory domain. Autophosphorylation is an intersubunit reaction occurring within each holoenzyme and requires that calmodulin activate one subunit serving as kinase, while a second calmodulin is bound to the subunit serving as substrate (4 -6). Autophosphorylation traps bound calmodulin by greatly reducing its dissociation rate and prolonging the active state (7). Even after calmodulin dissociates, the autophosphorylated kinase remains partially active or autonomous of Ca 2ϩ /calmodulin (8 -11). Phospho-Thr-286 may therefore be positioned to interfere with re-establishment of auto-inhibitory contacts. This autophosphorylation is critical for Ca 2ϩ spike frequency-dependent activation of the enzyme (12) and enhances targeting of the kinase to synaptic sites (13-15) that may underlie the role of the kinase in regulation of synaptic strength. Mice defective in this autophosphorylation (␣-CaM kinase II Thr-286 3 Ala mutants) do not exhibit long term potentiation and are defective in spatial learning (...

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Section: Discussionmentioning

confidence: 99%

Structural Examination of Autoregulation of Multifunctional Calcium/Calmodulin-dependent Protein Kinase II

Yang

Schulman

1999

Journal of Biological Chemistry

140

152

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“…Of the previously identified I1-associated LCs (LC8 and Tctex1), LC8 is apparently required for assembly because the LC8 null mutant (fla14) produces short flagella due to defects in intraflagellar transport (21) that have inner arm, outer arm, and radial spoke defects (LC8 is a component of all three complexes) (12,13,20). No tctex1 mutant has been identified in Chlamydomonas.…”

Section: Tctex2b Is Required For Dynein Motor Functionmentioning

confidence: 99%

“…Indeed, strains that lack various subsets of inner arms exhibit defects in waveform associated with reductions in shear amplitude (4). Subspecies f, or inner arm I1, contains 2 HCs 1 (1␣ and 1␤), three ICs (IC110, IC138, and IC140), and several LCs, including LC8 and Tctex1 (7)(8)(9)(10)(11)(12)(13). Both the 1␣ and 1␤ HCs are essential for the assembly of inner arm I1 (9,14), and a truncated 1␤ HC fragment containing the N-terminal ϳ113 kDa but lacking the motor domain is sufficient for this activity (14).…”

mentioning

confidence: 99%

A Novel Tctex2-related Light Chain Is Required for Stability of Inner Dynein Arm I1 and Motor Function in the Chlamydomonas Flagellum

DiBella

Smith

Patel‐King

et al. 2004

Journal of Biological Chemistry

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Tctex1 and Tctex2 were originally described in mice as putative distorters/sterility factors involved in the non-Mendelian transmission of t haplotypes. Subsequently, these proteins were found to be light chains of both cytoplasmic and axonemal dyneins. We have now identified a novel Tctex2-related protein (Tctex2b) within the Chlamydomonas flagellum. Tctex2b copurifies with inner arm I1 after both sucrose gradient centrifugation and anion exchange chromatography. Unlike the Tctex2 homologue within the outer dynein arm, analysis of a Tctex2b-null strain indicates that this protein is not essential for assembly of inner arm I1. However, a lack of Tctex2b results in an unstable dynein particle that disassembles after high salt extraction from the axoneme. Cells lacking Tctex2b swim more slowly than wild type and exhibit a reduced flagellar beat frequency. Furthermore, using a microtubule sliding assay we observed that dynein motor function is reduced in vitro. These data indicate that Tctex2b is required for the stability of inner dynein arm I1 and wild-type axonemal dynein function.

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“…It also associates with a wide variety of other proteins such as the proapoptotic factor Bim, rabies virus P protein, and Drosophila swallow [for a recent review, see King, 2002]. LC8 is highly conserved; for example, the Chlamydomonas and murine proteins are 89% identical [King and Patel-King, 1995]. In mammals, there are three closely related LC8 isoforms, although it is not yet clear whether they play distinct functional roles [Wilson et al, 2001].…”

Section: Lc8 Dimermentioning

confidence: 99%

Backbone dynamics of dynein light chains

King

2003

Cell Motil. Cytoskeleton

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The M = 8,000 and 11,000 Outer Arm Dynein Light Chains from Chlamydomonas Flagella Have Cytoplasmic Homologues

Cited by 183 publications

References 33 publications

Structural Examination of Autoregulation of Multifunctional Calcium/Calmodulin-dependent Protein Kinase II

Structural Examination of Autoregulation of Multifunctional Calcium/Calmodulin-dependent Protein Kinase II

A Novel Tctex2-related Light Chain Is Required for Stability of Inner Dynein Arm I1 and Motor Function in the Chlamydomonas Flagellum

Backbone dynamics of dynein light chains

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