Regulation Of Flagellar Length And Assembly In Chlamydomonas By LF4,a MAPK-related Kinase

Cilia and flagella are hair-like and microtubule-based organelles with specific size,widely distributed from unicellular organisms to higher complex eukaryotes,and are highly conservative in evolution,performing essential roles in the cell movement,signal sensing,cell proliferation and differentiation.Therefore,defects in ciliary structure,length,assembly or disassembly,resulting in a variety of human disease and developmental disorders,such as retinal degeneration,polycystic disease,skeletal abnormality and other multiorgan ciliopathies.The RCK family such as ICK,MAK,MOK,the members of mitogen-activated protein kinase(MAPK)superfamily,are known as key regulators of ciliogenesis.Among various ciliopathies,a severe neonatal lethal recessive disorder,endocrine-cerebroosteodysplasia(ECO)syndrome was caused by point mutation of ICK.LF4,the homologous protein of MOK in Chlamydomonas,is one of the earlist discovered kinases implicated in ciliary assembly and length regulation.However,in addition to genetic analysis,little is known about its cellular properties,molecular functions and regulation mechanisms.LF4 is localized both in the flagella and cell body with enrichment at the two basal bodies illustrated by super resolution microscopy.During ciliary assembly,the T159 site of LF4 kinase activation loop is constitutively phosphorylated and remains at the basal bodies.Furtherly,kinase inactivation,T159 phosphorylation site mutation or C-terminal non-kinase domain deletions alter the basal bodies localization of LF4,but fail to rescue the lf4-3 mutant(a null mutant),indicating that kinase activity and C terminal are essential for proper flagellar assembly.Intraflagellar transport(IFT)is a key regulator affecting the ciliary length.By live imaging of IFT tracking,we found that LF4 does not affect the velocities of IFT.Biochemistry analysis revealed that the loss of LF4 causes accumulation of IFT proteins in the cilia and reduces the phosphorylation of the kinesin-II subunit FLA8/KIF3 B,indicating that LF4 negatively regulates IFT proteins entry.Furthermore,we study the relationships of LF4 with LF1,LF2,LF3,LF5.Three of these proteins---LF1,LF2 and LF3 are found to form the length regulatory complex(LRC)together.Detection of phosphorylation antibodies indicated that the cell-cycle related kinase,LF2 and the novel protein LF3 are essential for LF4 phosphorylation,and LF4 may be the substrate of LF2.To further explore LF4 associated proteins,we used the proteomics analysis of LF4-HA immunoprecipitation and analysis of lf4 mutant comparative phosphorylation proteomics to provide new clues for cilia length regulation preliminary.In summary,LF4 is localized at the basal bodies and is a constitutively active kinase.It may be activated by LF2 and regulates IFT proteins entry by affecting the phosphorylation of FLA8,further performing important roles in ciliary length and assembly regulation.