A Regulatory Light Chain of Ciliary Outer Arm Dynein inTetrahymena thermophila

Søren Tvorup Christensen,Charles Guerra,Yuuko Wada,Tyson Valentin,Ruth Hogue Angeletti,Peter Satir,Toshikazu Hamasaki

doi:10.1074/jbc.m008412200

Søren Tvorup Christensen, Charles Guerra + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m008412200

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Abstract

Ciliary beat frequency is primarily regulated by outer arm dyneins (22 S dynein). Chilcote and Johnson (Chilcote, T. J., and Johnson, K. A. (1990) J. Biol. Chem. 256, 17257-17266) previously studied isolated Tetrahymena 22 S dynein, identifying a protein p34, which showed cAMP-dependent phosphorylation. Here, we characterize the molecular biochemistry of p34 further, demonstrating that it is the functional ortholog of the 22 S dynein regulatory light chain, p29, in Paramecium. p34, thiophosphorylated in isolated axonemes in the presence of cAMP, co-purified with 22 S dynein and not with inner arm dynein (14 S dynein). Isolated 22 S dynein containing phosphorylated p34 showed approximately 70% increase in in vitro microtubule translocation velocity compared with its unphosphorylated counterpart. Extracted p34 rebound to isolated 22 S dynein from either Tetrahymena or Paramecium but not to 14 S dynein from either ciliate. Binding of radiolabeled p34 to 22 S dynein was competitive with p29. Phosphorylated p34 was not present in axonemes isolated from a mutant lacking outer arms. Two-dimensional gel electrophoresis followed by phosphorimaging revealed at least five phosphorylated p34-related spots, consistent with multiple phosphorylation sites in p34 or perhaps multiple isoforms of p34. These new features suggest that a class of outer arm dynein light chains including p34 regulates microtubule sliding velocity and consequently ciliary beat frequency through phosphorylation.

Highlights

Cilia are ubiquitous cellular nanomachines, found in protists and multicellular eukaryotes, including man, whose repetitive beat depends on a microtubule-based cytoskeleton, powered by molecular motors, the outer and inner rows of dynein arms
We characterize the molecular biochemistry of p34 further, demonstrating that it is the functional ortholog of the 22 S dynein regulatory light chain, p29, in Paramecium. p34, thiophosphorylated in isolated axonemes in the presence of cAMP, co-purified with 22 S dynein and not with inner arm dynein (14 S dynein)
At 38 °C where outer arm dyneins are greatly reduced in the mutant, similar changes were seen at p78, p34 phosphorylation remained at control levels regardless of whether cAMP and/or Ca2ϩ were present

Summary

A Regulatory Light Chain of Ciliary Outer Arm Dynein in Tetrahymena thermophila*

256, 17257–17266) previously studied isolated Tetrahymena 22 S dynein, identifying a protein p34, which showed cAMP-dependent phosphorylation. Two-dimensional gel electrophoresis followed by phosphorimaging revealed at least five phosphorylated p34-related spots, consistent with multiple phosphorylation sites in p34 or perhaps multiple isoforms of p34 These new features suggest that a class of outer arm dynein light chains including p34 regulates microtubule sliding velocity and ciliary beat frequency through phosphorylation. Chilcote and Johnson [13] studied phosphorylation of isolated Tetrahymena 22 S dynein They were successful in phosphorylating 22 S dynein using the catalytic subunit of cAMP-dependent protein kinase from bovine heart with 500 ␮M ATP and vanadate as a phosphatase inhibitor.

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION