Characterization of a dual specificity protein phosphatase, MKP1, in Chlamydomonas

J.A. Buchheim1, Mark A. Buchheim2, and N.F. Wilson1

1) Oklahoma State University Center for Health Sciences, Tulsa, OK 74107, USA 2) University of Tulsa, Tulsa, OK, USA

Chlamydomonas actively maintains flagella that are equal and defined in length. The LF4 gene encodes a MAP kinase present in both cell bodies and flagella required for the regulation of flagellar length. MAP kinases require the phosphorylation of a threonine and tyrosine residue of a TXY motif within the kinase domain for enzyme activity. Previous studies revealed that LF4p was phosphorylated on the TEY motif in cell bodies but not in flagella. To identify negative regulators of LF4p enzyme activity in flagella, we examined the genome sequence for the presence of dual specificity protein phosphatases (DSPs) and identified eight DSPs. Northern blot analysis revealed that MKP1 is upregulated during flagellar regeneration suggesting this phosphatase could be a candidate for a negative regulator of LF4p enzyme activity. Immunoblot analysis demonstrated that MKP1 is present in cell bodies but not flagella. Similarly, immunofluorescent microscopy localized MKP1 to two discrete spots at the apical end of the cell body. Extraction of cell bodies with 0.5% NP-40 resulted in the release of ~25% of MKP1. The remaining MKP1 was not solubilized with higher detergent concentrations or with 0.6 M NaCl. Consistent with the immunolocalization of MKP1 to basal bodies, analysis of nuclear flagellar apparatuses revealed a enrichment in MKP1. Currently, we are employing isolation of nuclear flagellar apparatuses combined with immunofluorescent and immunogold electron microscopy to confirm the localization of MKP1 to basal bodies. Supported by OCAST grant HR-164.

e-mail address of presenting author: nedra.wilson@okstate.edu