Roles for a lipid phosphatase in the activation of its opposing lipid kinase

Abstract

Fig4 is a phosphoinositide phosphatase that converts PI3,5P2 to PI3P. Paradoxically, mutation of Fig4 results in lower PI3,5P2, indicating that Fig4 is also required for PI3,5P2 production. Fig4 promotes elevation of PI3,5P2, in part, through stabilization of a protein complex that includes its opposing lipid kinase, Fab1, and the scaffold protein Vac14. Here we show that multiple regions of Fig4 contribute to its roles in the elevation of PI3,5P2: its catalytic site, an N-terminal disease-related surface, and a C-terminal region. We show that mutation of the Fig4 catalytic site enhances the formation of the Fab1-Vac14-Fig4 complex, and loses some ability to elevate PI3,5P2. This suggests that independent of its lipid phosphatase function, the active site plays a role in the Fab1-Vac14-Fig4 complex. We also show that the N-terminal disease-related surface contributes to the elevation of PI3,5P2, and promotes Fig4 association with Vac14 in a manner that requires the Fig4 C-terminus. We find that the Fig4 C-terminus alone interacts with Vac14 in vivo, and retains some functions of full-length Fig4. Thus, a subset of Fig4 functions are independent of its phosphatase domain and at least three regions of Fig4 play roles in the function of the Fab1-Vac14-Fig4 complex.

Insights into Lysosomal PI(3,5)P2 Homeostasis from a Structural-Biochemical Analysis of the PIKfyve Lipid Kinase Complex

“…As another approach, we also overexpressed and purified the two versions of the complex in mammalian cells (Expi293F) and similarly analyzed PIKfyve phosphosites, with similar results (S48 and S2053 are differentially phosphorylated). These results strongly support the idea that, in addition to acting as PI(3,5)P 2 phosphatase, Fig4 is a serine phosphatase that regulates PIKfyve lipid kinase activity, explaining why an active form of Fig4 is required for normal levels of PI(3,5)P 2 in vivo (Duex et al, 2006a(Duex et al, , 2006bStrunk et al, 2020). PTEN, the phosphatase that dephosphorylates PI(3,4,5)P 3 during phosphatidylinositol 3-kinase (PI3K)/Akt signaling, also is active on protein substrates (Myers et al, 1997), setting a precedent for phosphatases with dual specificity on lipids and proteins.…”

“…This interaction slightly twists one of the Vac14 copies, likely modifying a surface on its opposite face to facilitate PIKfyve binding, consistent with PIKfyve's reported inability to bind Fig4 or Vac14 alone (Ikonomov et al, 2009). We did not observe density that could be attributed to portions of Fig4 downstream of its catalytic domain, although Fig4 C-terminal regions interact with Vac14 (Ikonomov et al, 2009;Strunk et al, 2020).…”

“…The observations (1) that PIKfyve is both a lipid and a protein kinase whose autophosphorylation affects both lipid kinase and phosphatase activities of the PIKfyve complex (Sbrissa et al, 2000 and above) and (2) that Fig4 catalytic activity is required for PIKfyve upregulation in vivo (Duex et al, 2006a(Duex et al, , 2006bStrunk et al, 2020) prompted us to explore the possibility that Fig4 might affect PIKfyve activity as a protein phosphatase. To determine whether Fig4 might act on PIKfyve, we prepared two versions of the PIKfyve/Fig4/Vac14 complex, as described before: one containing wild type and the other a catalytically inactivated Fig4 mutant (C486S).…”

“…How these activities are coordinated within the complex to avoid futile cycles of ATP hydrolysis has been enigmatic. Further mysterious is the observation that PI(3,5)P 2 levels in cells deficient in Fig4 activity, as, for example, in patients with CMT, are reduced rather than upregulated (Chow et al, 2007;Shisheva et al, 2019), as might have been expected, and that catalytically active Fig4 is required for PI(3,5)P 2 upregulation (Duex et al, 2006a(Duex et al, , 2006bStrunk et al, 2020).…”

A Compendium of Information on the Lysosome

“…Aside from its structural role in the complex and its lipid phosphatase activity, Fig4 is also a protein phosphatase targeting PIKfyve, priming the enzyme. This explains why catalytically active Fig4 is necessary for the activity of the complex ( Duex et al, 2006a ; Duex et al, 2006b ; Lees et al, 2020 ; Strunk et al, 2020 ). Furthermore, PIKfyve inhibits its lipid kinase activity by autophosphorylation which in turn stimulates Fig4 activity.…”