Ellen J. Tisdale

Rab2 Interacts Directly with Atypical Protein Kinase C (aPKC) ι/λ and Inhibits aPKCι/λ-dependent Glyceraldehyde-3-phosphate Dehydrogenase Phosphorylation

Atypical protein kinase C ι/λ (PKCι/λ) is essential for protein transport in the early secretory pathway. The small GTPase Rab2 selectively recruits the kinase to vesicular tubular clusters (VTCs) where PKCι/λ phosphorylates glyceraldehyde-3-phosphate dehydrogenase (GAPDH). VTCs are composed of small vesicles and tubules and serve as transport intermediates that shuttle cargo from the endoplasmic reticulum to the Golgi complex. These structures are the first site of segregation of the anterograde and retrograde pathways. When Rab2 binds to a VTC subcompartment, the subsequent recruitment of PKCι/λ and soluble components, including COPI (coatomer and ADP-ribosylation factor), results in the release of retrograde-directed vesicles. Because Rab2 stimulates PKCι/λ membrane association in a dose-dependent manner, we investigated whether the two proteins physically interact. Using a combination of in vivo and in vitro assays, we found that Rab2 interacts directly with PKCι/λ and that this interaction occurs through the Rab2 amino terminus (residues 1–19) and the PKCι/λ regulatory domain. A mutant lacking the PKCι/λ binding domain (Rab2N′Δ19) was functionally characterized. In contrast to Rab2, Rab2N′Δ19 failed to recruit PKCι/λ to normal rat kidney microsomes in a quantitative binding assay. To determine whether Rab2 modulates the ability of PKCι/λ to phosphorylate GAPDH, an in vitro kinase assay was supplemented with Rab2 or Rab2N′Δ19. Rab2 inhibited PKCι/λ-dependent GAPDH phosphorylation, whereas no effect was observed when the assay was performed with the aminoterminal truncation mutant. These results suggest that a downstream effector recruited to the VTC stimulates PKCι/λ-mediated GAPDH phosphorylation by alleviating the inhibition imposed by Rab2-PKCι/λ interaction.

A GAPDH Mutant Defective in Src-Dependent Tyrosine Phosphorylation Impedes Rab2-Mediated Events

Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) has multiple intracellular activities in addition to its role in gluconeogenesis. Indeed, we have reported that GAPDH is required for Rab2‐mediated retrograde transport from vesicular tubular clusters (VTCs). These diverse GAPDH activities are the result of posttranslational modifications that confer a new function to the enzyme. In that regard, GAPDH is tyrosine phosphorylated by Src. To establish the functional significance of this modification for GAPDH activity in Rab2‐dependent events, an amino acid substitution was made at tyrosine 41 (GAPDH Y41F). The inability of Src to phosphorylate purified recombinant GAPDH Y41F was confirmed in an in vitro kinase assay. The mutant was then employed in a quantitative membrane‐binding assay that measures Rab2 recruitment of soluble components to VTCs. As we observed with GAPDH wild type, Rab2 promoted GAPDH Y41F binding to membranes in a dose‐dependent manner, indicating that GAPDH tyrosine phosphorylation is not required for VTC association. However, GAPDH was tyrosine phosphorylated on VTCs. Importantly, GAPDH Y41F blocked vesicular stomatitis virus‐G transport in an assay that reconstitutes endoplasmic reticulum to Golgi trafficking, indicating that phosphorylation of tyrosine 41 is essential for GAPDH activity in the early secretory pathway. The block in transport is because of the decreased binding of atypical protein kinase C ι/λ to GAPDH Y41F, which reduces β‐coat protein association with the VTC and subsequent formation of Rab2‐mediated retrograde vesicles. Our results suggest that Src plays a pivotal role in regulating the interaction of Rab2 effectors on the VTC.

Rab2 Requires PKCι/Λ to Recruit β-COP for Vesicle Formation

The small GTPase Rab2 initiates the recruitment of soluble components necessary for protein sorting and recycling from pre‐Golgi intermediates. Our previous studies showed that Rab2 required protein kinase C (PKC) or a PKC‐like protein to recruit β‐COP to membrane (Tisdale EJ, Jackson M. Rab2 protein enhances coatomer recruitment to pre‐Golgi intermediates. J Biol Chem 1998;273: 17269–17277). We investigated the role of PKC in Rab2 function by first determining the active isoform that associates with membranes used in our assay. Western blot analysis detected three isoforms: PKCα, Γ and ι/Λ. A quantitative binding assay was used to measure recruitment of these kinases when incubated with Rab2. Only PKCι/Λ translocated to membrane in a dose‐dependent manner. Microsomes treated with anti‐PKCι/Λ lost the ability to bind β‐COP, suggesting that Rab2 requires PKCι/Λ for β‐COP recruitment. The recruitment of β‐COP to membranes is not regulated by PKCι/Λ kinase activity. However, PKCι/Λ kinase activity was necessary for Rab2‐mediated vesicle budding. We found that the addition of either a kinase‐deficient PKCι/Λ mutant or atypical PKC pseudosubstrate peptide to the binding assay drastically reduced vesicle formation. These data suggest that Rab2 causes translocation of PKCι/Λ to v esicular t ubular c lusters (VTCs), which promotes the recruitment of COPI to generate retrograde‐transport vesicles.

Class III PI 3-kinase is the main source of PtdIns3P substrate and membrane recruitment signal for PIKfyve constitutive function in podocyte endomembrane homeostasis

The evolutionarily conserved PIKfyve, which synthesizes PtdIns5P from PtdIns, and PtdIns(3,5)P2 from PtdIns3P, requires PtdIns3P as both an enzyme substrate and a membrane recruitment signal. Whereas the PtdIns3P source is undetermined, class III PI3K (Vps34), the only evolutionarily conserved of the eight mammalian PI3Ks, is presumed as a main candidate. A hallmark of PIKfyve deficiency is formation of multiple translucent cytoplasmic vacuoles seen by light microscopy in cells cultured in complete media. Such an aberrant phenotype is often observed in cells from conditional Vps34 knockout (KO) mice. To clarify the mechanism of Vps34 KO-triggered vacuolation and the PtdIns3P source for PIKfyve functionality, here we have characterized a podocyte cell type derived from Vps34fl/fl mice, which, upon Cre-mediated gene KO, robustly formed cytoplasmic vacuoles resembling those in PikfyveKO MEFs. Vps34wt, expressed in Vps34KO podocytes restored the normal morphology, but only if the endogenous PIKfyve activity was intact. Conversely, expressed PIKfyvewt rescued completely the vacuolation only in PikfyveKO MEFs but not in Vps34KO podocytes. Analyses of phosphoinositide profiles by HPLC and localization patterns by a PtdIns3P biosensor revealed that Vps34 is the main supplier of localized PtdIns3P not only for PIKfyve activity but also for membrane recruitment. Concordantly, Vps34KO podocytes had severely reduced steady-state levels of both PtdIns(3,5)P2 and PtdIns5P, along with PtdIns3P. We further revealed a plausible physiologically-relevant Vps34-independent PtdIns3P supply for PIKfyve, operating through activated class I PI3Ks. Our data provide the first evidence that the vacuolation phenotype in Vps34KO podocytes is due to PIKfyve dysfunction and that Vps34 is a main PtdIns3P source for constitutive PIKfyve functionality.

Atypical Protein Kinase C Plays a Critical Role in Protein Transport from Pre-Golgi Intermediates

The small GTPase Rab2 requires atypical protein kinase C ι/λ (PKCι/λ) kinase activity to promote vesicle budding from normal rat kidney cell microsomes (Tisdale, E. J. (2000) Traffic 1, 702–712). The released vesicles lack anterograde-directed cargo but contain coat protein I (COPI) and the recycling protein p53/p58, suggesting that the vesicles traffic in the retrograde pathway. In this study, we have directly characterized the role of PKCι/λ in the early secretory pathway. A peptide corresponding to the unique PKCι/λ pseudosubstrate domain was introduced into an in vitro assay that efficiently reconstitutes transport of vesicular stomatitis virus glycoprotein from the endoplasmic reticulum to the cis-medial Golgi compartments. This peptide blocked transport in a dose-dependent manner. Moreover, normal rat kidney cells incubated with Rab2 and the pseudosubstrate peptide displayed abundant swollen or dilated vesicles that contained Rab2, PKCι/λ, β-COP, and p53/p58. Because Rab2, β-COP, and p53/p58 are marker proteins for pre-Golgi intermediates (vesicular tubular clusters,VTCs), most probably the swollen vesicles are derived from VTCs. Similar results were obtained when the assays were supplemented with kinase-dead PKCι/λ (W274K). Both the pseudosubstrate peptide and kinase-dead PKCι/λ in tandem with Rab2 caused sustained membrane association of PKCι/λ, suggesting that reverse translocation was inhibited. Importantly, the inhibitory phenotype of kinase-dead PKCι/λ was reversed by PKCι/λ wild type. These combined results indicate that PKCι/λ is essential for protein transport in the early secretory pathway and suggest that PKCι/λ kinase activity is required to promote Rab2-mediated vesicle budding at a VTC subcompartment enriched in recycling cargo.

GAPDH binds Akt to facilitate cargo transport in the early secretory pathway

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) undergoes numerous post-translational modifications, which impart new function and influence intracellular location. For example, atypical PKC ι/λ phosphorylates GAPDH that locates to vesicular tubular clusters and is required for retrograde membrane trafficking in the early secretory pathway. GAPDH is also required in the endocytic pathway; substitution of Pro234 to Ser (Pro234Ser) rendered CHO cells defective in endocytosis. To determine if GAPDH (Pro234Ser) could inhibit endoplasmic reticulum to Golgi trafficking, we introduced the recombinant mutant enzyme into several biochemical and morphological transport assays. The mutant protein efficiently blocked vesicular stomatitis virus-G protein transport. Because GAPDH binds to microtubules (MTs), we evaluated MT binding and MT intracellular distribution in the presence of the mutant. Although these properties were not changed relative to wild-type, GAPDH (Pro234Ser) altered Golgi complex morphology. We determined that the GAPDH point mutation disrupted association between the enzyme and the serine/threonine kinase Akt. Interestingly Rab1, which functions in anterograde-directed trafficking, stimulates GAPDH-Akt association with membranes in a quantitative binding assay. In contrast, Rab2 does not stimulate GAPDH-Akt membrane binding but instead recruits GAPDH-aPKC. We propose a mechanism whereby the association of GAPDH with Akt or with aPKC serves as a switch to discriminate between anterograde directed cargo and recycling cargo retrieved back to the ER, respectively.

Overexpression of atypical protein kinase C in HeLa cells facilitates macropinocytosis via Src activation

Atypical protein kinase C (aPKC) is the first recognized kinase oncogene. However, the specific contribution of aPKC to cancer progression is unclear. The pseudosubstrate domain of aPKC is different from the other PKC family members, and therefore a synthetic peptide corresponding to the aPKC pseudosubstrate (aPKC-PS) sequence, which specifically blocks aPKC kinase activity, is a valuable tool to assess the role of aPKC in various cellular processes. Here, we learned that HeLa cells incubated with membrane permeable aPKC-PS peptide displayed dilated heterogeneous vesicles labeled with peptide that were subsequently identified as macropinosomes. A quantitative membrane binding assay revealed that aPKC-PS peptide stimulated aPKC recruitment to membranes and activated Src. Similarly, aPKC overexpression in transfected HeLa cells activated Src and induced macropinosome formation. Src-aPKC interaction was essential; substitution of the proline residues in aPKC that associate with the Src-SH3 binding domain rendered the mutant kinase unable to induce macropinocytosis in transfected cells. We propose that aPKC overexpression is a contributing factor to cell transformation by interacting with and consequently promoting Src activation and constitutive macropinocytosis, which increases uptake of extracellular factors, required for altered cell growth and accelerated cell migration.

The protein complex of neurodegeneration-related phosphoinositide phosphatase Sac3 and ArPIKfyve binds the Lewy body-associated synphilin-1, preventing its aggregation

Background: The cytosolic ArPIKfyve-Sac3 complex binds PIKfyve to regulate housekeeping endosomal functions but other tissue-specific interactors and functions are unknown. Results: Brain Synphilin-1 is a novel interaction partner of the ArPIKfyve-Sac3 complex. Conclusion: The ArPIKfyve-Sac3 complex is an effective inhibitor of aggregate formation by Synphilin-1. Significance: The novel molecular means for reducing cytoplasmic aggregates of Synphilin-1 provides new insights into neurodegeneration mechanisms. The 5-phosphoinositide phosphatase Sac3, in which loss-of-function mutations are linked to neurodegenerative disorders, forms a stable cytosolic complex with the scaffolding protein ArPIKfyve. The ArPIKfyve-Sac3 heterodimer interacts with the phosphoinositide 5-kinase PIKfyve in a ubiquitous ternary complex that couples PtdIns(3,5)P2 synthesis with turnover at endosomal membranes, thereby regulating the housekeeping endocytic transport in eukaryotes. Neuron-specific associations of the ArPIKfyve-Sac3 heterodimer, which may shed light on the neuropathological mechanisms triggered by Sac3 dysfunction, are unknown. Here we conducted mass spectrometry analysis for brain-derived interactors of ArPIKfyve-Sac3 and unraveled the α-synuclein-interacting protein Synphilin-1 (Sph1) as a new component of the ArPIKfyve-Sac3 complex. Sph1, a predominantly neuronal protein that facilitates aggregation of α-synuclein, is a major component of Lewy body inclusions in neurodegenerative α-synucleinopathies. Modulations in ArPIKfyve/Sac3 protein levels by RNA silencing or overexpression in several mammalian cell lines, including human neuronal SH-SY5Y or primary mouse cortical neurons, revealed that the ArPIKfyve-Sac3 complex specifically altered the aggregation properties of Sph1-GFP. This effect required an active Sac3 phosphatase and proceeded through mechanisms that involved increased Sph1-GFP partitioning into the cytosol and removal of Sph1-GFP aggregates by basal autophagy but not by the proteasomal system. If uncoupled from ArPIKfyve elevation, overexpressed Sac3 readily aggregated, markedly enhancing the aggregation potential of Sph1-GFP. These data identify a novel role of the ArPIKfyve-Sac3 complex in the mechanisms controlling aggregate formation of Sph1 and suggest that Sac3 protein deficiency or overproduction may facilitate aggregation of aggregation-prone proteins, thereby precipitating the onset of multiple neuronal disorders.