One of the challenges in understanding ciliary and flagellar motility is determining the mechanisms that locally regulate dynein-driven microtubule sliding. Our recent studies demonstrated that microtubule sliding, in Chlamydomonas flagella, is regulated by phosphorylation. However, the regulatory proteins remain unknown. Here we identify the 138-kD intermediate chain of inner arm dynein I1 as the critical phosphoprotein required for regulation of motility. This conclusion is founded on the results of three different experimental approaches. First, genetic analysis and functional assays revealed that regulation of microtubule sliding, by phosphorylation, requires inner arm dynein I1. Second, in vitro phosphorylation indicated the 138-kD intermediate chain of I1 is the only phosphorylated subunit. Third, in vitro reconstitution demonstrated that phosphorylation and dephosphorylation of the 138-kD intermediate chain inhibits and restores wild-type microtubule sliding, respectively. We conclude that change in phosphorylation of the 138-kD intermediate chain of I1 regulates dynein-driven microtubule sliding. Moreover, based on these and other data, we predict that regulation of I1 activity is involved in modulation of flagellar waveform.

IQGAP1 colocalizes with actin filaments in the cell cortex and binds in vitro to F-actin and several signaling proteins, including calmodulin, Cdc42, Rac1, and ␤-catenin. It is thought that the F-actin binding activity of IQGAP1 is regulated by its reversible association with these signaling molecules, but the mechanisms have remained obscure. Here we describe the regulatory mechanism for calmodulin. Purified adrenal IQGAP1 was found to consist of two distinct protein pools, one of which bound F-actin and lacked calmodulin, and the other of which did not bind F-actin but was tightly associated with calmodulin. Based on this finding we hypothesized that calmodulin negatively regulates binding of IQGAP1 to F-actin. This hypothesis was tested in vitro using recombinant wild type and mutated IQGAP1s and in live cells that transiently expressed IQGAP1-YFP. In vitro, the affinity of wild type IQGAP1 for F-actin decreased with increasing concentrations of calmodulin, and this effect was dramatically enhanced by Ca 2؉ and required the IQ domains of IQGAP1. In addition, we found that calmodulin bound wild type IQGAP1 much more efficiently in the presence of Ca Actin filament organization in the cell is regulated by a diverse set of factors that collectively control actin polymerization, actin filament length, interfilament cross-links, and interactions of polymerized actin with other cytoskeletal systems and membranes. One such regulatory factor,

(Eaf2) is a potential human tumor suppressor that exhibits frequent allelic loss and downregulation in high-grade prostate cancer. U19/Eaf2, along with its homolog Eaf1, has been reported to regulate transcriptional elongation via interaction with the eleven-nineteen lysine-rich leukemia (ELL) family of proteins. To further explore the tumor-suppressive effects of U19/Eaf2, we constructed and characterized a murine U19/Eaf2-knockout model. Homozygous or heterozygous deletion of U19/Eaf2 resulted in high rates of lung adenocarcinoma, B-cell lymphoma, hepatocellular carcinoma and prostate intraepithelial neoplasia. Within the mouse prostate, U19/Eaf2 deficiency enhanced cell proliferation and increased epithelial cell size. The knockout mice also exhibited cardiac cell hypertrophy. These data indicate a role for U19/Eaf2 in growth suppression and cell size control as well as argue for U19/Eaf2 as a novel tumor suppressor in multiple mouse tissues. The U19/Eaf2 knockout mouse also provides a unique animal model for three important cancers: lung adenocarcinoma, B-cell lymphoma and hepatocellular carcinoma.

Abstract. Genetic, biochemical, and structural data support a model in which axonemal radial spokes regulate dynein-driven microtubule sliding in Chlamydomonas flagella. However, the molecular mechanism by which dynein activity is regulated is unknown. We describe results from three different in vitro approaches to test the hypothesis that an axonemal protein kinase inhibits dynein in spoke-deficient axonemes from Chlamydomonas flagella. First, the velocity of dynein-driven microtubule sliding in spokedeficient mutants (pfl4, pfl7) was increased to wildtype level after treatment with the kinase-inhibitors HA-1004 or H-7 or by the specific peptide inhibitors of cAMP-dependent protein kinase (cAPK) PKI(6-22)amide or N~-acetyl-PKI(6-22)amide. In particular, the peptide inhibitors of cAPK were very potent, stimulating half-maximal velocity at 12-15 nM. In contrast, kinase inhibitors did not affect microtubule sliding in axonemes from wild-type cells. PKI treatment of axonemes from a double mutant missing both the radial spokes and the outer row of dynein arms (pf14pf28) also increased microtubule sliding to control (pf28) velocity. Second, addition of the type-II regulatory subunit of cAPK (RII) to spoke-deficient axonemes increased microtubule sliding to wild-type velocity. Addition of 10 #M cAMP to spokeless axonemes, reconstituted with RU, reversed the effect of RII. Third, our previous studies revealed that inner dynein arms from the Chlamydomonas mutants pf28 or pf14pf28 could be extracted in high salt buffer and subsequently reconstituted onto extracted axonemes restoring original microtubule sliding activity. Inner arm dyneins isolated from PKI-treated axonemes (mutam strain pf14pf28) generated fast microtubule sliding velocities when reconstituted onto both PKItreated or control axonemes. In contrast, dynein from control axonemes generated slow microtubule sliding velocities on either PKI-treated or control axonemes. Together, the data indicate that an endogenous axonemal cAPK-type protein kinase inhibits dynein-driven microtubule sliding in spoke-deficient axonemes. 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 outer dynein arms. The results are consistent with a model in which the radial spokes regulate dynein activity through suppression of a cAMP-mediated mechanism.YNEINS are a family of microtubule-based mechanoenzymes responsible for eukaryotic ciliary and flagellar motility, as well as for certain forms of cytoplasmic transport (Gibbons, 1989). Although much is known about the composition and structural organization of dyneins in flagellar axonemes (Witman, 1992), little is known about the coordinated regulation of dynein-driven microtubule sliding that produces regular bending of flagella (see Brokaw, 1994).

The inheritance of mitochondrial haplogroup U is associated with an approximately 2-fold increased risk of prostate cancer and 2.5-fold increased risk of renal cancer in white North American individuals. Therefore, individuals with this mitochondrial haplotype are in a high risk group. Because mitochondrial haplogroup U is found in 9.35% of the white United States population, there are more than 20 million individuals in this high risk group.