Molecular Basis for Association of PIPKIγ-p90 with Clathrin Adaptor AP-2

Abstract

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ) is an essential determinant in clathrin-mediated endocytosis (CME). In mammals three type I phosphatidylinositol-4-phosphate 5-kinase (PIPK) enzymes are expressed, with the I␥-p90 isoform being highly expressed in the brain where it regulates synaptic vesicle (SV) exo-/endocytosis at nerve terminals. How precisely PI(4,5)P 2 metabolism is controlled spatially and temporally is still uncertain, but recent data indicate that direct interactions between type I PIPK and components of the endocytic machinery, in particular the AP-2 adaptor complex, are involved. Here we demonstrated that PIPKI␥-p90 associates with both the and ␤2 subunits of AP-2 via multiple sites. Crystallographic data show that a peptide derived from the splice insert of the human PIPKI␥-p90 tail binds to a cognate recognition site on the sandwich subdomain of the ␤2 appendage. Partly overlapping aromatic and hydrophobic residues within the same peptide also can engage the C-terminal sorting signal binding domain of AP-2, thereby potentially competing with the sorting of conventional YXXØ motif-containing cargo. Biochemical and structure-based mutagenesis analysis revealed that association of the tail domain of PIPKI␥-p90 with AP-2 involves both of these sites. Accordingly the ability of overexpressed PIPKI␥ tail to impair endocytosis of SVs in primary neurons largely depends on its association with AP-2␤ and AP-2. Our data also suggest that interactions between AP-2 and the tail domain of PIPKI␥-p90 may serve to regulate complex formation and enzymatic activity. We postulate a model according to which multiple interactions between PIPKI␥-p90 and AP-2 lead to spatiotemporally controlled PI(4,5)P 2 synthesis during clathrin-mediated SV endocytosis.Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ) 5 plays a key role in a variety of cell physiological pathways including but not limited to CME, neurotransmitter release (31) and SV cycling, phagocytosis, cell signaling and proliferation, and regulation of the actin cytoskeleton, as well as nuclear functions (1-7). In mammals these diverse roles of PI(4,5)P 2 are regulated by only three type I phosphatidylinositol-4-phosphate 5-kinase (PIPK) isozymes. Although PIPKI␣ and -␤ are ubiquitous proteins, the type I␥ isozyme is most highly expressed in the brain where it localizes to synaptic sites (8). PIPKI␥ is also present at focal adhesion sites in non-neuronal cells (9, 10).An overwhelming body of data implicates PI(4,5)P 2 in CME and in the exo-/endocytic cycling of SV membranes (2-4). Recruitment of clathrin adaptors, including epsins, CALM/ AP180, Dab2, HIP1/1R, and AP-2, to the plasma membrane strictly requires the presence of PI(4,5)P 2 , which associates directly with cognate recognition motifs and domains within these proteins. AP-2 is a heterotetrameric complex composed of ␣, ␤2, 2, and 2 subunits that fold into a largely ␣-helical solenoid (termed the "core") with two appendage domains joined to the core by long flexible linkers (11). Membrane...

PIPKIγi5 regulates the endosomal trafficking and degradation of E-cadherin

“…PIPKIci2 and PIPKIci5 are expressed in the majority of epithelial and nonepithelial cell lines, where they have unique functions. PIPKIci2 regulates the trafficking of several proteins to and from the plasma membrane (Bairstow et al, 2006;Kahlfeldt et al, 2010;Ling et al, 2007). The function of PIPKIci5 is still emerging.…”

“…Specific isoforms have distinct functions. For example, PIPKIci2 regulates focal adhesion dynamics and vesicle trafficking (Di Paolo et al, 2002;Kahlfeldt et al, 2010;Ling et al, 2002). In epithelial cells, PIPKIci2 regulates the formation of cell-cell contacts through its association with the adhesion molecule E-cadherin and a specific interaction with the epithelial-specific AP1B clathrin adaptor complex .…”

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

“…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.…”

“…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).…”

“…The two major splice isoforms of PIPKIc, encoded by the Pip5k1c gene in mouse, PIPKIc_i1 (also known as PIPKIc635 or PIPKIc87) and PIPKIc_i2 (also known as PIPKIc661 or PIPKIc90) localize to cell-cell junctions of epithelial cells (Akiyama et al, 2005) or focal adhesions (Di Paolo et al, 2002;Ling et al, 2002), respectively. They differ by the addition of 26 amino acids to the C-terminus of PIPKIc_i2, that provides a binding site for the clathrin AP-2 adaptor complex b2 and m subunits, and the integrin adaptor talin (de Pereda et al, 2005;Kahlfeldt et al, 2010).…”