Clathrin Regulates the Association of PIPKIγ661 with the AP-2 Adaptor β2 Appendage

Phosphoinositides are spatially restricted membrane signaling molecules. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), a phosphoinositide highly enriched in and present throughout the plasma membrane, has been implicated in endocytosis. Trypanosoma brucei has one of the highest known rates of endocytosis, a process it uses to evade the immune system. To determine whether phosphoinositides play a role in endocytosis in this organism we have identified and characterized one of the enzymes responsible for generating PI(4,5)P2. Surprisingly, this phosphoinositide was found to be highly concentrated in the flagellar pocket, the only site of endocytosis and exocytosis in this organism. The enzyme (designated TbPIPKA, annotated as Tb927.10.1620), furthermore, was present at the neck of the pocket, towards the anterior end of the parasite. Depletion of TbPIPKA led to depletion of PI(4,5)P2 and enlargement of the pocket, the result of impaired endocytosis. Together, these data suggest that TbPIPKA and its product PI(4,5)P2 are important for endocytosis and consequently for flagellar pocket homeostasis.

Section: Introductionmentioning

confidence: 99%

The endocytic activity of the flagellar pocket in Trypanosoma brucei is regulated by an adjacent phosphatidylinositol phosphate kinase

Demmel

Schmidt

Lucast

et al. 2014

“…PIPKIc isoforms are targeted to distinct subcellular sites. By regulating the dynamics of regional PI(4,5)P 2 pools, they are implicated in distinct cellular processes such as vesicular trafficking (Bairstow et al, 2006;Ling et al, 2007;Sun et al, 2013;Thapa et al, 2012;Thieman et al, 2009;Xiong et al, 2012), calcium signaling (Wang et al, 2004) or cell adhesion and migration (Di Paolo et al, 2002;El Sayegh et al, 2007;Ling et al, 2007a;Ling et al, 2002;Sun et al, 2007;Thapa et al, 2012;Thieman et al, 2009;Wang et al, 2004;Xiong et al, 2012). Moreover, PIPKIc can regulate some proteins, such as E-cadherin , by directly interacting with them.…”

Section: Introductionmentioning

confidence: 99%

Type Iγ phosphatidylinositol phosphate kinase targets to the centrosome and restrains centriole duplication

Zhang

Xiong

et al. 2014

Self Cite

Centriole biogenesis depends on the Polo-like kinase PLK4 and a small group of structural proteins. The spatiotemporal regulation of these proteins at pre-existing centrioles is critical to ensure that centriole duplication occurs once per cell cycle. Here we report that type Iγ phosphatidylinositol-4-phosphate 5-kinase (PIPKIγ) plays an important role in centriole fidelity. Depending upon an association with CEP152, PIPKIγ localized in a ring-like pattern in the intermediate pericentriolar materials around the proximal end of the centriole in G1, S, and G2 phases, but not in M phase. Without detaining cells in S or M phase, depletion of PIPKIγ led to centriole amplification in a PLK4/SAS-6 dependent manner. Expression of exogenous PIPKIγ reduced centriole amplification resulted from endogenous PIPKIγ depletion, hydroxyurea treatment, or PLK4 overexpression, suggesting that PIPKIγ likely functions at the PLK4 level to restrain centriole duplication. Importantly, we found that PIPKIγ bound to the cryptic Polo-Box domain of PLK4 and this binding reduced PLK4 kinase activity. Together, our findings suggest that PIPKIγ is a novel negative regulator of centriole duplication by modulating the homeostasis of PLK4 activity.

“…Analysis of these mice revealed a reduction in PtdIns(4,5)P 2 levels in brain accompanied by synaptic vesicle (SV) recycling defects. As PIPKIc interfaces with the clathrin-endocytosis machinery via interactions between the AP-2 adaptor complex and either the Cterminal extension present in PIPKIc_i2 (Bairstow et al, 2006;Nakano-Kobayashi et al, 2007;Thieman et al, 2009;Kahlfeldt et al, 2010) or the kinase domain (Krauss et al, 2006) we sought to confirm the findings in our PIPKIc 2/2 mouse and extend them to the PIPKIcDE17 mouse.…”

Section: Endocytosis Defects In Pipkicmentioning

confidence: 56%

“…PIPKIc_i2 was shown to be the relevant splice isoform in neurons (Nakano-Kobayashi et al, 2007) and was also shown to be an important mediator of endocytosis in MDCK and HeLa cells (Bairstow et al, 2006). Mutational and structural analysis indicates that the 26 amino acid extension of PIPKIc_i2 binds to the m2 subunit of the AP-2 adaptor complex (Bairstow et al, 2006;Kahlfeldt et al, 2010), as well as to the b2 subunit (Nakano- Kobayashi et al, 2007;Thieman et al, 2009;Kahlfeldt et al, 2010), thereby localizing PIPKIc_i2 to clathrin-coated pits. However, the kinase core domain of all PIPKIs also binds to m2, raising the possibility that other PIPKIs and splice isoforms of PIPKIc may stimulate endocytosis in some cell types (Krauss et al, 2006).…”

Section: Discussionmentioning

confidence: 99%

Comparative phenotypic analysis of the two major splice isoforms of phosphatidylinositol phosphate kinase type Iγ in vivo

Legate

Montag

Böttcher

et al. 2012

Localized production of polyphosphoinositides is critical for their signaling function. To examine the biological relevance of specific pools of phosphatidylinositol 4,5-bisphosphate we compared the consequences of genetically ablating all isoforms of PIP kinase Type Iγ (PIPKIγ) versus ablation of a specific splice isoform, PIPKIγ_i2, with respect to three reported PIPKIγ functions. Ablation of PIPKIγ_i2 caused a neuron-specific endocytosis defect similar to that found in PIPKIγ −/− mice, while agonist-induced calcium signaling was blunted in PIPKIγ −/− cells, but was not affected in the absence of PIPKIγ_i2. A reported contribution of PIPKIγ to epithelial integrity was not evident in PIPKIγ −/− mice. As mice lacking PIPKIγ_i2 live a normal lifespan whereas PIPKIγ −/− mice die shortly after birth, we propose that PIPKIγ-mediated metabotropic calcium signaling may represent an essential function of PIPKIγ, whereas functions specific to the PIPKIγ_i2 splice isoform are not essential for survival.