PIKfyve regulation

PIKfyve may be phosphorylated on a number of different, some of which have a regulatory function. This post starts with a quick overview of PIKfyve interacting with the chaperone Vac14 that also binds the phosphatase Fig4. Regulation of PIKfyve is way more complicated that this. One of many phosphorylation sites was chosen for closer examination.

PIKfyve is a kinase which has two functions

• phosphorylation of phosphatidylinositol on the fifth hydroxyl of the myo-inositol ring, to form phosphatidylinositol 5-phosphate (PtdIns₅P), a controller of membrane trafficking.
• phosphorylation of phosphatidylinositol 3-phosphate on the fifth hydroxyl of the myo-inositol ring, to form PtdIns(3,5)P₂, a phospholipid of endosome fission and fusion.

This post starts with a quick overview of PIKfyve interacting with the chaperon Vac14 that also binds the phosphatase Fig4. Regulation of PIKfyve is way more complicated that this. One of many phosphorylation sites was chosen for closer examination.

Phosphatase and Kinase in one complex [1,2]

This is a recycled image from a previous post on chloride transport in lysosomes. The kinase PIKFYVE generates the signaling lipid PI(3,5)P₂ on the surface of endosomes and lysosomes. PIKfyve production of PI(3,5)P₂ inhibits CIC7. [1] PIKjyve can also attach a phosphate group to itself. Fig4 can take this phosphate off PIKkyve and PI(3,5)P₂ . It is way more complicated in that has many PI(3,5)P₂ regulatory functions.

Mutations in CLCN7, gain or loss of function?

Many CLCN7 mutations occur in the PI(3,5)P₂ binding site. There is an ongoing discussion of over acidification, alkalinization, and just loss of fine tuning of hydrolase activity in response to the environment.

the crystal structure [2]

Lees and coworkers have published an excellent structure of PIKfyve (the stapler), Fig4 (the staple remover), and Vac14 (the hand). [2] Instead of putting a staple in a substrate PIKfyve inserts a phosphate group. It can insert a phosphate on itself. It is hard enough to imagine having a stapler and staple remove in one hand! Fig4 is of source the staple remover. In this analogy it can not only be stapled, but it can remove the staple from itself.

Now imagine that the staple’s activity is regulated by where the samples are. The stapler can staple itself as well s receive stables from other staples.

Phosphorylation of PIKfyve

Before we get to caught up with overly academic papers we have to remember that enzymes in our bodies are subject to fine tuning with “post translational modifications.” Phosphorylation, attachment of a phosphate group to serine, threonine, or tyrosine, is perhaps the most common modification to proteins after they are translated from messenger RNA. Uniprot has two databases on all known phosphorylation sites on PIKfyve. The PRIDE database is quite extensive and does not reference peer reviewed publication The UniProt list of phosphorylation sites is presented on this post.One of these appears to be particularly promising in terms of lysosome function is ULK phosphorylation .

according to PRIDE

Proteomics Identification Database (PRIDE) is a UK site in which users submit sequences of peptides. After the submission, the dataset metadata is validated and different post-processing are applied. PRIDE Submission Tool enables direct user submissions of protein and peptide identification or quantification data, along with the spectra to PRIDE Archive. Note that combined sources, not just one, point to phosphorylation of numerous serine and threonine phosphorylation sites.

according to UnProt

The Universal Protein Resource database of phosphorylation sites appears to be a bit smaller. A nice thing about this database is that it often contains information as which kinase is responsible for the phosphorylation.

This post is not going to get into the experimental details of the Karabiyik 2021 publication [3] Suffice it to say that glucose deprivation can activate at least two protein kinases that activate PIKfyve.

A few of many regulatory sites on PIKfyve [3]

The UniProt entry for PIKfyve is 1541 MDASPRNISP 1550. S is Serine, one of the amino acids subject to phosphorylation. “ULK1 activated by AMPK during glucose starvation phosphorylates the lipid kinase PIKfyve on S1548, thereby increasing its activity and the synthesis of the phospholipid PI(5)P without changing the levels of PI(3,5)P₂. ULK1-mediated activation of PIKfyve enhances the formation of PI(5)P-containing autophagosomes upon glucose starvation, resulting in an increase in autophagy flux. Phospho-mimic PIKfyve S1548D drives autophagy upregulation and lowers autophagy substrate levels. Our study has identified how ULK1 upregulates autophagy upon glucose starvation and induces the formation of PI(5)P-containing autophagosomes by activating PIKfyve.”

Click for the graphical abstract of Karabiyik 2021. Garphical abstracts are figures that summarize the entire study. Figure 6 has an addendum. Showing that phosphorylated (activated) AMPK will phosphorylate PIKfyve on serine 307 thus activating phosphorylation of PI(3)P to PI(3,5)P₂, a phospholipid in the endocytic pathway, possibly of amyloid deposits in Alzheimer’s Disease.

Note that this phosphorylation is not producing the inhibitor of the CIC7 Cl^–/H⁺ antiporter PI(3,5)P₂

Wolfberries, a Traditional Chinese Medicine treatment[4]?

The goal of this study was to test the hypothesis that wolfberries could prevent hepatic steatosis in mice fed a high fat diet. Wild type mice were compared with AMPKα2 knockout mice. The α subunit is one of three in AMP kinase. Wolfberries contain the carotenoids zeaxanthin and lutein. Somehow these carotenoids counter the free fatty acid inactivation of AMPKα2 that leads to decreased mitochondrial biogenesis and suppressed mitophagy.

References

1. Cao X, Lenk GM, Mikusevic V, Mindell JA, Meisler MH. The chloride antiporter CLCN7 is a modifier of lysosome dysfunction in FIG4 and VAC14 mutants. PLoS Genet. 2023 Jun 26;19(6):e1010800. PMC free article
2. Lees JA, Li P, Kumar N, Weisman LS, Reinisch KM. Insights into Lysosomal PI(3,5)P₂ Homeostasis from a Structural-Biochemical Analysis of the PIKfyve Lipid Kinase Complex. Mol Cell. 2020 Nov 19;80(4):736-743.e4. PMC free article
3. Karabiyik C, Vicinanza M, Son SM, Rubinsztein DC. Glucose starvation induces autophagy via ULK1-mediated activation of PIKfyve in an AMPK-dependent manner. Dev Cell. 2021 Jul 12;56(13):1961-1975.e5. PMC free article
4. Lin D, He H, Ji H, Willis J, Willard L, Jiang Y, Medeiros DM, Wark L, Han J, Liu Y, Lu B. Wolfberries potentiate mitophagy and enhance mitochondrial biogenesis leading to prevention of hepatic steatosis in obese mice: the role of AMP-activated protein kinase α2 subunit. Mol Nutr Food Res. 2014 May;58(5):1005-15. Sci-Hub free article