Biological and Pathological Implications of an Alternative ATP-Powered Proteasomal Assembly With Cdc48 and the 20S Peptidase

The ATP-powered protein degradation machinery plays essential roles in maintaining protein homeostasis in all organisms. Robust proteolytic activities are typically sequestered within protein complexes to avoid the fatal removal of essential proteins. Because the openings of proteolytic chambers are narrow, substrate proteins must undergo unfolding. AAA superfamily proteins (ATPases associated with diverse cellular activities) are mostly located at these openings and regulate protein degradation appropriately. The 26S proteasome, comprising 20S peptidase and 19S regulatory particles, is the major ATP-powered protein degradation machinery in eukaryotes. The 19S particles are composed of six AAA proteins and 13 regulatory proteins, and bind to both ends of a barrel-shaped proteolytic chamber formed by the 20S peptidase. Several recent studies have reported that another AAA protein, Cdc48, can replace the 19S particles to form an alternative ATP-powered proteasomal complex, i.e., the Cdc48-20S proteasome. This review focuses on our current knowledge of this alternative proteasome and its possible linkage to amyotrophic lateral sclerosis

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

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

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

Identification of Nedd4 as a Kif26b-interacting protein.

<p><i>A</i>. HEK293 cells were transfected with the indicated constructs. FLAG-tagged proteins were immunoprecipitated from the lysates with anti-FLAG beads. The precipitates were analyzed by immunoblotting with the indicated antibodies. <i>B</i>. Schematic diagrams of the Kif26b deletion constructs. Interaction with Nedd4 is summarized on the <i>right</i> of each construct: +, positive; −, negative. <i>C</i>. HEK293 cells were transfected with the indicated constructs. FLAG-tagged proteins were immunoprecipitated from the lysates with anti-FLAG beads. The precipitates were analyzed by immunoblotting with the indicated antibodies. <i>D</i>. Schematic diagrams of the Nedd4 deletion constructs. Interaction with Kif26b is summarized on the right of each construct: +, positive; −, negative. <i>E</i>. HEK293 cells were transfected with FLAG-Kif26b-expressing plasmids. The lysates were incubated with purified GST or GST-tagged Nedd4 deletions. The glutathione bead precipitates were analyzed by immunoblotting with the indicated antibodies.</p

Phosphorylation and polyubiquitination of Kif26b in the developing kidney.

<p><i>A</i>. E14.5 kidneys were cultured <i>in vitro</i> for 8 h with or without roscovitine (20 µM) (indicated as “+” or “−,” respectively). Kif26b was immunoprecipitated from the lysates with anti-Kif26b antibody and subjected to immunoblotting with the indicated antibodies. Immunoprecipitations with rabbit IgG and without lysate were performed as controls. <i>B</i>. Kidneys dissected from E14.5 embryos were stained with the indicated antibodies. Arrow heads indicate the metanephric mesenchyme. Scale bar = 10 µm. <i>C.</i> Kif26b was immunoprecipitated from E14.5 kidney lysates with anti-Kif26b antibody, and precipitates were treated with or without AP for 1 h at 37°C and analyzed by immunoblotting with the indicated antibodies (left panel). E14.5 kidneys were cultured <i>in vitro</i> for 8 h with or without roscovitine (20 µM) (indicated as “+” or “−,” respectively). Kif26b was immunoprecipitated and subjected to immunoblotting with the indicated antibodies (right panel). <i>D.</i> E14.5 kidneys were cultured <i>in vitro</i> for 8 h with or without MG132 (20 µM) (indicated as “+” or “−,” respectively). Kif26b was immunoprecipitated from the lysates with anti-Kif26b antibody and subjected to immunoblotting with the indicated antibodies.</p

Kif26b interacts with Nedd4 in a phosphorylation-dependent manner.

<p><i>A</i>. HEK293 cells were transfected with the indicated constructs. At 48 h after post-transfection, cells were treated for 6 h with DMSO (-) or Roscovitine (+; 20 µM) and then lysed, and the FLAG-tagged proteins were immunoprecipitated with anti-FLAG beads. The precipitates were analyzed by immunoblotting with the indicated antibodies. <i>B.</i> HEK293 cells were transfected with the indicated constructs. 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