REGULATION OF FLAGELLAR DYNEIN BY AN AXONEMAL TYPE-1 PHOSPHATASE IN CHLAMYDOMONAS

Physiological studies have demonstrated that flagellar radial spokes r egulate inner arm dynein activity in Chlamydomonas and that an axonema l cAMP-dependent kinase inhibits dynein activity in radial spoke defec tive axonemes, These studies also suggested that an axonemal protein p hosphatase is required for activation of flagellar dynein, We tested w hether inhibitors of protein phosphatases would prevent activation of dynein by the kinase inhibitor PKI in Chlamydomonas axonemes lacking r adial spokes, As predicted, preincubation of spoke defective axonemes (pf14 and pf17) with ATP gamma S maintained the slow dynein-driven mic rotubule sliding characteristic of paralyzed axonemes lacking spokes, and blocked activation of dynein-driven microtubule sliding by subsequ ent addition of PKI. Preincubation of spoke defective axonemes with th e phosphatase inhibitors okadaic acid, microcystin-LR or inhibitor-2 a lso potently blocked PKI-induced activation of microtubule sliding vel ocity: the non-inhibitory okadaic acid analog, 1-norokadaone, did not, ATP gamma S or the phosphatase inhibitors blocked activation of dynei n in a double mutant lacking the radial spokes and the outer dynein ar ms (pf14pf28). We concluded that the axoneme contains a type-1 phospha tase required for activation of inner arm dynein. We postulated that t he radial spokes regulate dynein through the activity of the type-1 pr otein phosphatase, To test this, we performed in vitro reconstitution experiments using inner arm dynein from the double mutant pf14pf28 and dynein-depleted axonemes containing wild-type radial spokes (pf28), A s described previously, microtubule sliding velocity was increased fro m similar to 2 mu m/second to similar to 7 mu m/second when inner arm dynein from pf14pf28 axonemes was reconstituted with axonemes containi ng wild-type spokes. In contrast, pretreatment of inner arm dynein fro m pf14pf28 axonemes with ATP gamma S, or reconstitution in the presenc e of microcystin-LR, blocked increased velocity following reconstituti on, despite the presence of wild-type radial spokes, We conclude that the radial spokes, through the activity of an axonemal type-1 phosphat ase, activate inner arm dynein by dephosphorylation of a critical dyne in component. Wild-type radial spokes also operate to inhibit the axon emal cAMP-dependent kinase, which would otherwise inhibit axonemal dyn ein and motility.