Based on motif searches, it was determined that there are two predicted tyrosine phosphorylation sites in Kelch. The first is in the BTB dimerization domain at residue 132, and the second is within the fifth kelch repeat (KREP) at residue 627. The fifth KREP in alpha-scruin has been shown to bind F-actin (Sun, 1997). Site-directed mutagenesis was performed to change the tyrosines to alanines, which are incapable of being phosphorylated. Transgenes encoding these constructs were designated P[kelY132A] and P[kelY627A]. At least three transgenic lines for each mutant were examined in a kelch mutant background (Kelso, 2002).
To examine Kelch phosphorylation in the Drosophila ovary, 2D electrophoresis of ovary lysates was performed from several genetic backgrounds. When wild-type ovary lysates were treated with phosphatase inhibitors, a tyrosine-phosphorylated protein that comigrated with one of two Kelch isoforms was detected. Antibodies to phosphoserine and phosphothreonine showed no immunoreactivity comigrating with Kelch. Comparison to pH standards shows that the shift observed between the two Kelch isoforms is equivalent to the addition of a single phosphate. In the absence of phosphatase inhibitors, Kelch migrates as a single spot with no corresponding phosphotyrosine staining. Egg chambers dissected from src64Delta17 homozygous flies do not contain the phosphorylated form of Kelch. In the presence of phosphatase inhibitors, Kelch protein from kelDE1;P[kelY132A]/+ ovaries continues to be tyrosine phosphorylated. However, Kelch tyrosine phosphorylation in kelDE1;P[kelY627A]/+ ovaries is absent. To characterize the effects of phosphorylation on the ability of Kelch to bind actin, an actin overlay experiment was performed. Total ovary lysates from wild-type ovaries were separated using 2D electrophoresis, and the blots were incubated with F-actin and then actin antibodies to detect bound actin. Only nonphosphorylated Kelch (KelY627A) binds actin. To verify that actin binding was due to Kelch, ovary lysates from a kelch mutant were tested; there was no longer actin binding present in the area where Kelch protein would have focused. These results indicate that the tyrosine residue at position 627 is phosphorylated in a Src64-dependent manner, and phosphorylation of that residue disrupts Kelch binding to actin (Kelso, 2002).
The ability of purified recombinant Kelch protein to interact with F-actin was tested. The majority of Kelch sediments in the presence of F-actin and remains in the supernatant in its absence. Saturation binding was determined by incubating purified Kelch with phalloidin-stabilized F-actin in increasing ratios, followed by 100,000 g centrifugation. As the Kelch/actin ratio increases, more Kelch remaines in the supernatant. The stoichiometry of Kelch binding to actin was estimated using a saturation curve in which the ratio of Kelch to actin in the pellet was plotted against increasing concentrations of Kelch. From this curve, the molar ratio of Kelch to F-actin was calculated as 1:4 (Kelso, 2002).
To determine if Kelch cross-links F-actin, low speed sedimentation at 16,000 g was performed. When a saturating concentration of Kelch is added, most of the F-actin forms pellets when centrifuged, suggesting that the actin is being cross-linked into bundles. Negative staining of F-actin, sedimented with or without Kelch, was performed to further determine the nature of the interaction. The actin-alone sample contains mainly single filaments. In contrast, pellets from mixtures of Kelch and actin contain mostly loose bundles of actin filaments. These observations suggest that full-length Kelch acts to bundle ring canal F-actin (Kelso, 2002).
The F-actin binding domain was mapped to a single KREP by expressing and purifying each individual repeat and then performing high speed centrifugation in the presence of F-actin. KREPs 1-4 and 6 fail to cosediment with F-actin. KREP five is capable of binding F-actin in a saturable manner. To address the effect of phosphorylation, aspartate or glutamate residues were introduced at position 627 to mimic phosphorylation. Both substitutions disrupt the ability of KREP five to bind F-actin, resulting in >50% of each mutant repeat remaining in the supernatant after high speed centrifugation. To demonstrate that the substitution made in the KelY627A protein does not affect actin binding, the substitution was tested in vitro. Neither alanine nor phenylalanine substitutions disrupt actin binding. These data suggest that F-actin binding by KREP five is likely to be reduced by phosphorylation of the tyrosine residue at position 627 (Kelso, 2002).
To study the role of Kelch phosphorylation in vivo, kelch mutant flies expressing KelY627A protein in the germline were examined. Wild-type stage 10A egg chambers stained with the Kel1B monoclonal antibody have a ring canal staining pattern that colocalizes with F-actin. Egg chambers from kelch mutants show a complete absence of Kelch protein staining, and the well-characterized phenotype of ring canal actin disorganization with partial occlusion of the lumen (Tilney, 1996; Robinson, 1997a). The expression of one copy of P[kelY627A] in kelch mutants results in restoration of Kelch localization to ring canals and a rescue of F-actin organization, showing that the KelY627A protein has F-actin binding and cross-linking activity in vivo. The phenotype of KelY627A is not changed in a src64Delta17 background. At a higher magnification, horizontal sections of wild-type ring canals are characterized by the appearance of two parallel rims of actin. In kelch mutants, there is the typical collapse of actin into the lumen, almost completely obstructing the ring canal. Comparison of wild type, kelDE1;P[kelY627A]/+, and src64Delta17 reveals an increase in the concavity of the actin rim in both mutants, causing the appearance of a bicycle rim shape as well as a decrease in the diameter of the ring canal. These observations suggest that the phenotype in kelDE1;P[kelY627A] closely resembles src64Delta17. Additionally, the introduction of two copies of P[kelY627A] into a wild-type background causes a decrease in ring canal diameter. This dominant-negative phenotype indicates that the introduction of 'irreversible' cross-links formed by KelY627A perturbs the function of endogenous Kelch (Kelso, 2002).
Many studies have focused on roles for Src family kinases (SFKs) in regulation of proliferation, differentiation and dynamic changes in cellular morphology. In this report, Src64 is shown to be dispensable for proliferation and differentiation of both germ cells and follicle cells in the Drosophila ovary. Instead, Src64 is required for morphological changes at the ring canal and contributes to the packaging of germline cysts by follicle cells during egg chamber formation. The results demonstrate that Csk regulates Src64 function during packaging, but is dispensable during ring canal growth control. Thus, regulation of Src64 activity levels during these two morphological events is distinct (O'Reilly, 2006).
Actin polymerization is a crucial component of ring canal growth regulation, and mutation of genes that control actin dynamics causes dramatic ring canal defects. Src64Delta17 ring canals are smaller than wild type and exhibit diminished actin polymerization. Recent work has shown that Src64-mediated phosphorylation of the actin-bundling protein Kelch is crucial for regulating actin polymerization during ring canal growth. Whereas the Src64Delta17 ring canal defects are strikingly similar to those observed in germ cells expressing only [Kelch YA], which cannot be tyrosyl phosphorylated by Src64, it was found that Src64KO ring canal growth defects are more severe than those in Src64Delta17 or, by inference, [KelchYA] mutants. This result suggests that Src64 may control additional signals during this process. Cortactin or members of the WASP/SCAR protein family promote actin polymerization through Arp2/3 complex activation and are required for ring canal growth regulation. Both types of protein are known vertebrate SFK substrates, suggesting the possibility that several Src64-dependent routes may drive the actin polymerization required for ring canal growth (O'Reilly, 2006).
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