Phosphorylation of Kif26b Promotes Its Polyubiquitination and Subsequent Proteasomal Degradation during Kidney Development

Kif26b, a member of the kinesin superfamily proteins (KIFs), is essential for kidney development. Kif26b expression is restricted to the metanephric mesenchyme, and its transcription is regulated by a zinc finger transcriptional regulator Sall1. However, the mechanism(s) by which Kif26b protein is regulated remain unknown. Here, we demonstrate phosphorylation and subsequent polyubiquitination of Kif26b in the developing kidney. We find that Kif26b interacts with an E3 ubiquitin ligase, neural precursor cell expressed developmentally down-regulated protein 4 (Nedd4) in developing kidney. Phosphorylation of Kif26b at Thr-1859 and Ser-1962 by the cyclin-dependent kinases (CDKs) enhances the interaction of Kif26b with Nedd4. Nedd4 polyubiquitinates Kif26b and thereby promotes degradation of Kif26b via the ubiquitin-proteasome pathway. Furthermore, Kif26b lacks ATPase activity but does associate with microtubules. Nocodazole treatment not only disrupts the localization of Kif26b to microtubules but also promotes phosphorylation and polyubiquitination of Kif26b. These results suggest that the function of Kif26b is microtubule-based and that Kif26b degradation in the metanephric mesenchyme via the ubiquitin-proteasome pathway may be important for proper kidney development

Par1b/MARK2 Phosphorylates Kinesin-Like Motor Protein GAKIN/KIF13B To Regulate Axon Formation ▿ †

Here we report that Par1b/MARK2 regulates axon formation via phosphorylation of a kinesin superfamily protein GAKIN/KIF13B. Accumulating evidence indicated the importance of the evolutionarily conserved kinase Par1b in the regulation of cell polarity. Using hippocampal neurons in culture, it has been shown that Par1b regulates axon specification, but the underlying mechanism remains uncharacterized. We identify GAKIN/KIF13B as a novel Par1b-binding protein and reveal that GAKIN/KIF13B is a physiological substrate for Par1b, and the phosphorylation sites are conserved from Drosophila. In hippocampal neurons, GAKIN/KIF13B accumulates at the distal part of the microtubules in the tips of axons, but not of dendrites. Overexpression of GAKIN/KIF13B by itself can induce the formation of extra axons, which is inhibited by the coexpression of Par1b in a manner dependent on its kinase activity. In contrast, small interfering RNA (siRNA)-mediated knockdown of GAKIN/KIF13B severely retards neurite extension and promotes the axonless phenotype. The extra axon phenotype caused by Par1b siRNA is suppressed by cointroduction of GAKIN/KIF13B siRNA, thus placing the GAKIN/KIF13B function downstream of Par1b. We also find that GAKIN/KIF13B acts downstream of the phosphatidylinositol 3-kinase (PI3K) signaling via Par1b phosphorylation. These results reveal that GAKIN/KIF13B is a key intermediate linking Par1b to the regulation of axon formation

The Tumor Suppressor MIG6 Controls Mitotic Progression and the G2/M DNA Damage Checkpoint by Stabilizing the WEE1 Kinase

MIG6 is an important tumor suppressor that binds to and negatively regulates epidermal growth factor receptor (EGFR). Here, we report an EGFR-independent function for MIG6 as an integral component of the cell cycle machinery. We found that depletion of MIG6 causes accelerated entry into and delayed exit from mitosis. This is due to premature and prolonged activation of CDK1, a key regulator of mitotic progression at the G2/M and meta- and anaphase transitions. Furthermore, MIG6 is required for inhibition of CDK1 upon DNA damage and subsequent G2/M cell cycle arrest. Mechanistically, we found that MIG6 depletion results in reduced phosphorylation of CDK1 on the inhibitory WEE1-targeted tyrosine-15 residue. MIG6 interacts with WEE1 and promotes its stability by interfering with the recruitment of the βTrCP-SCF E3 ubiquitin ligase and consequent proteasomal degradation of WEE1. Our findings uncover a critical role of MIG6 in cell cycle progression that is likely to contribute to its potent tumor-suppressive properties

Ubiquitination of Kif26b is promoted by Nedd4.

<p><i>A</i>. HEK293 cells were transfected with the indicated plasmids. At 48 h after post-transfection, the cells were treated for 8 h with MG132 (20 µM) and the FLAG-tagged proteins were then immunoprecipitated from the cell lysates with anti-FLAG beads. The precipitates were analyzed by immunoblotting with the indicated antibodies. <i>B</i>. HEK293 cells were transfected with FLAG-Kif26b. The FLAG-tagged proteins were immunoprecipitated from the cell lysates with anti-FLAG M2 beads, and the precipitates were used for <i>in vitro</i> ubiquitination assays with GST or GST-Nedd4 proteins (wild-type [WT] or catalytically inactive [CS]). The reaction mixtures were subjected to immunoblotting with the indicated antibodies. <i>C</i>. HEK293 cells were transfected with the indicated plasmids. At 48 h after post-transfection, the cells were pretreated for 3 h with DMSO or Roscovitine (20 µM) and then further treated for 8 h with MG132 (20 µM). The FLAG-tagged proteins were then immunoprecipitated from the cell lysates with anti-FLAG beads and subjected to immunoblotting with the indicated antibodies. <i>D</i>. HEK293 cells were transfected with the indicated plasmids. At 48 h after post-transfection, the cells were treated as in <i>A</i> and then the FLAG-tagged proteins were immunoprecipitated from the cell lysates with anti-FLAG beads and subjected to immunoblotting with the indicated antibodies. <i>E</i>. HeLa cells stably expressing FLAG-Kif26b were transfected with control or Nedd4-siRNAs. At 48 h after post-transfection, the cells were treated as in <i>A</i> and the FLAG-tagged proteins were then immunoprecipitated from the cell lysates with anti-FLAG beads. The precipitates were analyzed by immunoblotting with the indicated antibodies.</p

Phosphorylation and polyubiquitination of Kif26b are induced by disruption of microtubules.

<p><i>A.</i> HeLa cells stably expressing FLAG-Kif26b were pretreated for 3 h with DMSO or Roscovitine (20 µM) and then treated for the indicated time with 5 µM nocodazole (left panel). HEK293 cells stably expressing FLAG-Kif26b were pretreated for 3 h with DMSO or Roscovitine (20 µM) and then treated for 6 h with 5 µM nocodazole (right panel). The lysates were subjected to immunoblotting with the indicated antibodies. <i>B.</i> HeLa cells stably expressing FLAG-Kif26b were treated for 6 h with nocodazole (5 µM), and then the FLAG-tagged proteins were immunoprecipitated from the cell lysates with anti-FLAG beads. The precipitates were subsequently treated with or without AP at 37°C for 1 h and analyzed by immunoblotting with anti-FLAG antibody. <i>C</i>. HeLa or HEK293 cells stably expressing FLAG-Kif26b (left and right panel, respectively) were treated as in <i>B,</i> and then the FLAG-tagged proteins were immunoprecipitated from the cell lysates with anti-FLAG beads. The precipitates were analyzed by immunoblotting with the indicated antibodies. <i>D</i>. HeLa or HEK293 cells stably expressing FLAG-Kif26b (left and right panel, respectively) were pretreated for 6 h with nocodazole and then further treated for 8 h with MG132 (20 µM). The FLAG-tagged proteins were immunoprecipitated from the lysates with anti-FLAG beads and subjected to immunoblotting with the indicated antibodies. <i>E</i>. HeLa or HEK293 cells stably expressing FLAG-Kif26b (left and right panel, respectively) were treated for 8 h with DMSO, MG132 (20 µM), or chloroquine (100 µM). Whole cell lysates were subjected to SDS-PAGE followed by immunoblotting with the indicated antibodies.</p

Kif26b is an unconventional kinesin.

<p><i>A.</i> Alignment of the amino acid sequences of the motor domains of various KIFs. Human KIF5A, GAKIN/KIF13B, KIF26A, and KIF26B and mouse Kif26b are shown. Amino acids that correspond to the p-loop, L9-loop, and L11-loop consensus sequences are shown on a gray background. <i>B.</i> Schematic diagram of the microtubule-binding assay procedure (left panel). COS-7 cells were transfected with FLAG-Kif26b- or FLAG-GAKIN/KIF13B-expressing plasmids. At 48 h after post-transfection, the microtubule-binding assay was performed on the cell lysates and the precipitates were analyzed by immunoblotting with anti-FLAG antibody (right panel). <i>C.</i> HeLa cells stably expressing FLAG-Kif26b were treated for 1 h with DMSO or nocodazole (5 µM). The cells were then fixed and stained with anti-FLAG (green) and anti-β-tubulin (red) antibodies. Scale bar = 10 µm.</p

CDKs phosphorylate Thr-1859 and Ser-1962 on Kif26b.

<p><i>A.</i> Schematic diagram of the amino acids in Kif26b potentially phosphorylated by CDK5. The C-terminal tail region of Kif26b that interacts with Nedd4 is indicated by the bar. Underlined amino acids indicate CDK5 phosphorylation sites. <i>B.</i> GST or GST-Kif26b-C was incubated with or without recombinant His-tagged CDK1, CDK2, CDK5 or CDK6 and a kinase assay was performed. Proteins were separated by SDS-PAGE and detected by Coomassie Brilliant Blue (CBB) staining or immunoblotting with the indicated antibodies. <i>C</i>. HEK293 cells were transfected with a FLAG-Kif26b-expressing plasmid. At 48 hrs post-transfection, the cells were treated for 6 h with DMSO (-) or Roscovitine (+; 20 µM). FLAG-tagged proteins were immunoprecipitated from the cell lysates with anti-FLAG beads. Precipitates were then treated with or without calf intestine-derived alkaline phosphatase (AP) at 37°C for 1 h and analyzed by immunoblotting with the indicated antibodies. <i>D</i>. HEK293 cells were transfected with WT or substitution mutant FLAG-Kif26b-expressing plasmids. The FLAG-tagged proteins were immunoprecipitated from the cell lysates with anti-FLAG beads. The precipitates were analyzed by immunoblotting with the indicated antibodies.</p

A Coated Vesicle-associated Kinase of 104 kDa (CVAK104) Induces Lysosomal Degradation of Frizzled 5 (Fzd5)*

Receptor internalization is recognized as an important mechanism for controlling numerous cell surface receptors. This event contributes not only to regulate signal transduction but also to adjust the amount of cell surface receptors. Frizzleds (Fzds) are seven-pass transmembrane receptor family proteins for Wnt ligands. Recent studies indicated that Fzd5 is internalized in response to Wnt stimulation to activate downstream signaling pathways. After internalization, it appears that Fzd5 is recycled back to the plasma membrane. However, whether internalized Fzd5 is sorted to lysosomes for protein degradation remains unclear. We here report that a coated vesicle-associated kinase of 104 kDa (CVAK104) selectively induces lysosomal degradation of Fzd5. We identify CVAK104 as a novel binding partner of Dishevelled (Dvl), a scaffold protein in the Wnt signaling pathway. Interestingly, we find that CVAK104 also interacts with Fzd5 but not with Fzd1 or Fzd4. CVAK104 selectively induces intracellular accumulation of Fzd5 via the clathrin-mediated pathway, which is suppressed by coexpression of a dominant negative form of Rab5. Fzd5 is subsequently degraded by a lysosomal pathway. Indeed, knockdown of endogenous CVAK104 by RNA interference results in an increase in the amount of Fzd5. In contrast, Wnt treatment induces Fzd5 internalization but does not stimulate its degradation. Overexpression or knockdown of CVAK104 results in a significant suppression or activation of the Wnt/β-catenin pathway, respectively. These results suggest that CVAK104 regulates the amount of Fzd5 by inducing lysosomal degradation, which probably contributes to the suppression of the Wnt signaling pathway