REGULATION OF CHLAMYDOMONAS FLAGELLAR DYNEIN BY AN AXONEMAL PROTEIN-KINASE

Genetic, biochemical, and structural data support a model in which axo nemal radial spokes regulate dynein-driven microtubule sliding in Chla mydomonas flagella. However, the molecular mechanism by which dynein a ctivity is regulated is unknown. We describe results from three differ ent in vitro approaches to test the hypothesis that an axonemal protei n kinase inhibits dynein in spoke-deficient axonemes from Chlamydomona s flagella. First, the velocity of dynein-driven microtubule sliding i n spoke-deficient mutants (pf14, pf17) was increased to wildtype level after treatment with the kinase inhibitors HA-1004 or H-7 or by the s pecific peptide inhibitors of cAMP-dependent protein kinase (cAPK) PKI (6-22)amide or N-alpha-acetyl-PKI(6-22)amide. In particular, the pepti de inhibitors of cAPK were very potent, stimulating half-maximal veloc ity at 12-15 nM. In contrast, kinase inhibitors did not affect microtu bule sliding in axonemes from wild-type cells. PKI treatment of axonem es from a double mutant missing both the radial spokes and the outer r ow of dynein arms (pf14pf28) also increased microtubule sliding to con trol (pf28) velocity. Second, addition of the type-II regulatory subun it of cAPK (RII) to spoke-deficient axonemes increased microtubule sli ding to wild-type velocity. Addition of 10 mu M cAMP to spokeless axon emes, reconstituted with RII, reversed the effect of RII. Third, our p revious studies revealed that inner dynein arms from the Chlamydomonas mutants pf28 or pf14pf28 could be extracted in high salt buffer and s ubsequently reconstituted onto extracted axonemes restoring original m icrotubule sliding activity. Inner arm dyneins isolated from PKI-treat ed axonemes (mutant strain pf14pf28) generated fast microtubule slidin g velocities when reconstituted onto both PKI-treated or control axone mes. In contrast, dynein from control axonemes generated slow microtub ule sliding velocities on either PKI-treated or control axonemes. Toge ther, the data indicate that an endogenous axonemal cAPK-type protein kinase inhibits dynein-driven microtubule sliding in spoke-deficient a xonemes. The kinase is likely to reside in close association with its substrate(s), and the substrate targets are not exclusively localized to the central pair, radial spokes, dynein regulatory complex, or oute r dynein arms. The results are consistent with a model in which the ra dial spokes regulate dynein activity through suppression of a cAMP-med iated mechanism.