Characterization of the flagellar form of LF4p, a MAP kinase involved in the regulation of flagellar length
Nedra F. Wilson1,2, J.A. Buchheim2, K. Pargeter2, William D. Meek2, and Paul A. Lefebvre1
1) Department of Plant Biology; Univ. of Minnesota; St. Paul, MN 55108, USA
2) Department of Anatomy and Cell Biology; Oklahoma State Univ. Center for Health Sciences; Tulsa, OK 74107, USA
Chlamydomonas actively maintains flagella at an equal and appropriate length. Deletion of a MAP kinase, LF4, results in cells that are unable to regulate the length of flagella. LF4p is present in both cell bodies and flagella with the cell body form migrating as a doublet of 73/72 kDa and the cell body form migrating as a single 68 kDa form. Immunoblot analysis suggests both forms of LF4p within cell bodies are phosphorylated within the TXY motif found in the activation loop of MAP kinases. The flagellar form, however, was not recognized with the anti-pTEpY antibody. This lack of phosphorylation of the flagellar form of LF4p is consistent with in-gel kinase assays suggesting that in full-length steady state flagella, LF4p is largely enzymatically inactive. Examination of flagella of fla10, a temperature-sensitive mutant of the anterograde motor protein for intraflagellar transport (IFT), demonstrated the loss of LF4p at the restrictive temperature. Conversely, the level of LF4p is elevated in flagella of other long flagella mutants, lf1, lf2, and lf3. As we have been unable to localize LF4p by immunofluorescence, we utilized biochemical fraction of flagella. Immunoblot analysis of the resulting fractions revealed that LF4p is a component of the membrane + matrix fraction and was co-precipitated with a 30 kDa protein identified as 14-3-3. Several observations indicate that LF4p activity may regulate the levels of FLA10p in the flagellum. First, ultrastructural analysis of lf4 flagellar cross-sections demonstrated an approximate three-fold increase in the number of IFT particles relative to wild-type flagella. Second, lf4 flagella contain greatly increased amounts of FLA10p and KAP (the kinesin II accessory protein) relative to wild-type flagella. Supported by OCAST grant HR-164 and NIH GM034437.
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