Andrew C. Hedman

Phosphatidylinositol-4,5-bisphosphate: Targeted Production and Signaling

Phosphatidylinositol 4,5-bisphosphate (PI4,5P(2)) is a key lipid signaling molecule that regulates a vast array of biological activities. PI4,5P(2) can act directly as a messenger or can be utilized as a precursor to generate other messengers: inositol trisphosphate, diacylglycerol, or phosphatidylinositol 3,4,5-trisphosphate. PI4,5P(2) interacts with hundreds of different effector proteins. The enormous diversity of PI4,5P(2) effector proteins and the spatio-temporal control of PI4,5P(2) generation allow PI4,5P(2) signaling to control a broad spectrum of cellular functions. PI4,5P(2) is synthesized by phosphatidylinositol phosphate kinases (PIPKs). The array of PIPKs in cells enables their targeting to specific subcellular compartments through interactions with targeting factors that are often PI4,5P(2) effectors. These interactions are a mechanism to define spatial and temporal PI4,5P(2) synthesis and the specificity of PI4,5P(2) signaling. In turn, the regulation of PI4,5P(2) effectors at specific cellular compartments has implications for understanding how PI4,5P(2) controls cellular processes and its role in diseases.

Endosomal Type Iγ PIP 5-Kinase Controls EGF Receptor Lysosomal Sorting

Endosomal trafficking and degradation of epidermal growth factor receptor (EGFR) play an essential role in the control of its signaling. Phosphatidylinositol-4,5-bisphosphate (PtdIns4,5P(2)) is an established regulator of endocytosis, whereas PtdIns3P modulates endosomal trafficking. However, we demonstrate here that type I gamma phosphatidylinositol phosphate 5-kinase i5 (PIPKIγi5), an enzyme that synthesizes PtdIns4,5P(2), controls endosome-to-lysosome sorting of EGFR. In this pathway, PIPKIγi5 interacts with sorting nexin 5 (SNX5), a protein that binds PtdIns4,5P(2) and other phosphoinositides. PIPKIγi5 and SNX5 localize to endosomes, and loss of either protein blocks EGFR sorting into intraluminal vesicles (ILVs) of the multivesicular body. Loss of ILV sorting greatly enhances and prolongs EGFR signaling. PIPKIγi5 and SNX5 prevent Hrs ubiquitination, and this facilitates the Hrs association with EGFR that is required for ILV sorting. These findings reveal that PIPKIγi5 and SNX5 form a signaling nexus that controls EGFR endosomal sorting, degradation, and signaling.

IQGAP1 is a novel phosphatidylinositol 4,5 bisphosphate effector in regulation of directional cell migration

Phosphatidylinositol 4,5 bisphosphate (PIP2) is a key lipid messenger for regulation of cell migration. PIP2 modulates many effectors, but the specificity of PIP2 signalling can be defined by interactions of PIP2‐generating enzymes with PIP2 effectors. Here, we show that type Iγ phosphatidylinositol 4‐phosphate 5‐kinase (PIPKIγ) interacts with the cytoskeleton regulator, IQGAP1, and modulates IQGAP1 function in migration. We reveal that PIPKIγ is required for IQGAP1 recruitment to the leading edge membrane in response to integrin or growth factor receptor activation. Moreover, IQGAP1 is a PIP2 effector that directly binds PIP2 through a polybasic motif and PIP2 binding activates IQGAP1, facilitating actin polymerization. IQGAP1 mutants that lack PIPKIγ or PIP2 binding lose the ability to control directional cell migration. Collectively, these data reveal a synergy between PIPKIγ and IQGAP1 in the control of cell migration.

LAPTM4B is a PtdIns(4,5)P2 effector that regulates EGFR signaling, lysosomal sorting, and degradation

Lysosomal degradation is essential for the termination of EGF‐stimulated EGF receptor (EGFR) signaling. This requires EGFR sorting to the intraluminal vesicles (ILVs) of multi‐vesicular endosomes (MVEs). Cytosolic proteins including the ESCRT machineries are key regulators of EGFR intraluminal sorting, but roles for endosomal transmembrane proteins in receptor sorting are poorly defined. Here, we show that LAPTM4B, an endosomal transmembrane oncoprotein, inhibits EGF‐induced EGFR intraluminal sorting and lysosomal degradation, leading to enhanced and prolonged EGFR signaling. LAPTM4B blocks EGFR sorting by promoting ubiquitination of Hrs (an ESCRT‐0 subunit), which inhibits the Hrs association with ubiquitinated EGFR. This is counteracted by the endosomal PIP kinase, PIPKIγi5, which directly binds LAPTM4B and neutralizes the inhibitory function of LAPTM4B in EGFR sorting by generating PtdIns(4,5)P2 and recruiting SNX5. PtdIns(4,5)P2 and SNX5 function together to protect Hrs from ubiquitination, thereby promoting EGFR intraluminal sorting. These results reveal an essential layer of EGFR trafficking regulated by LAPTM4B, PtdIns(4,5)P2 signaling, and the ESCRT complex and define a mechanism by which the oncoprotein LAPTM4B can transform cells and promote tumor progression.

PIP kinases define PI4,5P2 signaling specificity by association with effectors ☆

Phosphatidylinositol 4,5-bisphosphate (PI4,5P₂) is an essential lipid messenger with roles in all eukaryotes and most aspects of human physiology. By controlling the targeting and activity of its effectors, PI4,5P₂modulates processes, such as cell migration, vesicular trafficking, cellular morphogenesis, signaling and gene expression. In cells, PI4,5P₂has a much higher concentration than other phosphoinositide species and its total content is largely unchanged in response to extracellular stimuli. The discovery of a vast array of PI4,5P₂ binding proteins is consistent with data showing that the majority of cellular PI4,5P₂is sequestered. This supports a mechanism where PI4,5P₂functions as a localized and highly specific messenger. Further support of this mechanism comes from the de novo synthesis of PI4,5P₂which is often linked with PIP kinase interaction with PI4,5P₂effectors and is a mechanism to define specificity of PI4,5P₂signaling. The association of PI4,5P₂-generating enzymes with PI4,5P₂effectors regulate effector function both temporally and spatially in cells. In this review, the PI4,5P₂effectors whose functions are tightly regulated by associations with PI4,5P₂-generating enzymes will be discussed. This article is part of a Special Issue entitled Phosphoinositides.

Isoform 5 of PIPKIγ regulates the endosomal trafficking and degradation of E-cadherin

ABSTRACT Phosphatidylinositol phosphate kinases (PIPKs) have distinct cellular targeting, allowing for site-specific synthesis of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] to activate specific signaling cascades required for cellular processes. Several C-terminal splice variants of PIPKI&ggr; (also known as PIP5K1C) exist, and have been implicated in a multitude of cellular roles. PI(4,5)P2 serves as a fundamental regulator of E-cadherin transport, and PI(4,5)P2-generating enzymes are important signaling relays in these pathways. We present evidence that the isoform 5 splice variant of PIPKI&ggr; (PIPKI&ggr;i5) associates with E-cadherin and promotes its lysosomal degradation. Additionally, we show that the endosomal trafficking proteins SNX5 and SNX6 associate with PIPKI&ggr;i5 and inhibit PIPKI&ggr;i5-mediated E-cadherin degradation. Following HGF stimulation, activated Src directly phosphorylates PIPKI&ggr;i5. Phosphorylation of the PIPKI&ggr;i5 C-terminus regulates its association with SNX5 and, consequently, E-cadherin degradation. Additionally, this PIPKI&ggr;i5-mediated pathway requires Rab7 to promote degradation of internalized E-cadherin. Taken together, the data indicate that PIPKI&ggr;i5 and SNX5 are crucial regulators of E-cadherin sorting and degradation. PIPKI&ggr;i5, SNX and phosphoinositide regulation of lysosomal sorting represent a novel area of PI(4,5)P2 signaling and research. PIPKI&ggr;i5 regulation of E-cadherin sorting for degradation might have broad implications in development and tissue maintenance, and enhanced PIPKI&ggr;i5 function might have pathogenic consequences due to downregulation of E-cadherin.

Phosphatidylinositol Phosphate 5-Kinase Iγi2 in Association with Src Controls Anchorage-independent Growth of Tumor Cells

Background: PIPKIγ isoforms play roles in cell migration, polarization, and membrane trafficking and are overexpressed in triple-negative breast cancers, indicating protumorigenic functions. Results: PIPKIγi2 associates with the C terminus of Src, and this interaction interdependently controls their functioning. Conclusion: PIPKIγi2 and Src synergistically control anchorage-independent tumor cell growth. Significance: This study shows unexpected mechanisms for a phosphatidylinositol 4,5-biphosphate-generating enzyme that synergizes with the proto-oncogene Src to regulate oncogenic signaling. A fundamental property of tumor cells is to defy anoikis, cell death caused by a lack of cell-matrix interaction, and grow in an anchorage-independent manner. How tumor cells organize signaling molecules at the plasma membrane to sustain oncogenic signals in the absence of cell-matrix interactions remains poorly understood. Here, we describe a role for phosphatidylinositol 4-phosphate 5-kinase (PIPK) Iγi2 in controlling anchorage-independent growth of tumor cells in coordination with the proto-oncogene Src. PIPKIγi2 regulated Src activation downstream of growth factor receptors and integrins. PIPKIγi2 directly interacted with the C-terminal tail of Src and regulated its subcellular localization in concert with talin, a cytoskeletal protein targeted to focal adhesions. Co-expression of PIPKIγi2 and Src synergistically induced the anchorage-independent growth of nonmalignant cells. This study uncovers a novel mechanism where a phosphoinositide-synthesizing enzyme, PIPKIγi2, functions with the proto-oncogene Src, to regulate oncogenic signaling.

An unexpected role for PI4,5P2 in EGF receptor endosomal trafficking

Epidermal growth factor receptor (EGFR) is highly expressed or overly active in many types of cancer, including head and neck, breast, ovarian, esophageal and non-small cell lung cancers.1 Increased expression of EGFR has been associated with resistance to standard therapies and poor patient prognosis. EGFR signaling promotes cancer progression by stimulating angiogenesis, cancer cell proliferation, migration, invasion and metastasis. To design more efficient anti-EGFR therapies for cancers, the mechanisms by which EGFR expression and signaling are modulated must be well defined.

Calmodulin Lobes Facilitate Dimerization and Activation of Estrogen Receptor-α

Estrogen receptor α (ER-α) is a nuclear hormone receptor that controls selected genes, thereby regulating proliferation and differentiation of target tissues, such as breast. Gene expression controlled by ER-α is modulated by Ca2+ via calmodulin (CaM). Here we present the NMR structure of Ca2+-CaM bound to two molecules of ER-α (residues 287–305). The two lobes of CaM bind to the same site on two separate ER-α molecules (residues 292, 296, 299, 302, and 303), which explains why CaM binds two molecules of ER-α in a 1:2 complex and stabilizes ER-α dimerization. Exposed glutamate residues in CaM (Glu-11, Glu-14, Glu-84, and Glu-87) form salt bridges with key lysine residues in ER-α (Lys-299, Lys-302, and Lys-303), which is likely to prevent ubiquitination at these sites and inhibit degradation of ER-α. Transfection of cells with full-length CaM slightly increased the ability of estrogen to enhance transcriptional activation by ER-α of endogenous estrogen-responsive genes. By contrast, expression of either the N- or C-lobe of CaM abrogated estrogen-stimulated transcription of the estrogen responsive genes pS2 and progesterone receptor. These data suggest that CaM-induced dimerization of ER-α is required for estrogen-stimulated transcriptional activation by the receptor. In light of the critical role of ER-α in breast carcinoma, our data suggest that small molecules that selectively disrupt the interaction of ER-α with CaM may be useful in the therapy of breast carcinoma.