PTEN is a tumor suppressor that reverses the action of phosphoinositide 3-kinase by catalyzing the removal of the 3 phosphate of phosphoinositides. Despite the critical role of PTEN in cell signaling and regulation, the mechanisms of its membrane recruitment and activation is still poorly understood. PTEN is composed of an N-terminal phosphatase domain, a C2 domain, and a C-terminal tail region that contains the PSD-95͞Dlg͞ZO-1 homology (PDZ) domain-binding sequence and multiple phosphorylation sites. Our in vitro surface plasmon resonance measurements using immobilized vesicles showed that both the phosphatase domain and the C2 domain, but not the C-terminal tail, are involved in electrostatic membrane binding of PTEN. Furthermore, the phosphorylationmimicking mutation on the C-terminal tail of PTEN caused an Ϸ80-fold reduction in its membrane affinity, mainly by slowing the membrane-association step. Subcellular localization studies of PTEN transfected into HEK293T and HeLa cells indicated that targeting of PTEN to the plasma membrane is coupled with rapid degradation and that the phosphatase domain and the C2 domain are both necessary and sufficient for its membrane recruitment. Results also indicated that the phosphorylation regulates the targeting of PTEN to the plasma membrane not by blocking the PDZ domain-binding site but by interfering with electrostatic membrane binding of PTEN. On the basis of these results, we propose a membrane-binding and activation mechanism for PTEN, in which the phosphorylation͞dephosphorylation of the C-terminal region serves as an electrostatic switch that controls the membrane translocation of the protein.

Highlights d Patched structure reveals a hydrophobic conduit with sterollike contents d Patched mediates Hedgehog-reversible reduction of inner leaflet cholesterol activity d Hydrophobic conduit is essential for cholesterol effect and Smoothened suppression d Inner leaflet cholesterol likely mediates Hedgehog-Patched regulation of Smoothened

The roles of cationic, aliphatic, and aromatic residues in the membrane association and dissociation of five phospholipases A(2) (PLA(2)), including Asp-49 PLA(2) from the venom of Agkistrodon piscivorus piscivorus, acidic PLA(2) from the venom of Naja naja atra, human group IIa and V PLA(2)s, and the C2 domain of cytosolic PLA(2), were determined by surface plasmon resonance analysis. Cationic interfacial binding residues of A. p. piscivorus PLA(2) (Lys-10) and human group IIa PLA(2) (Arg-7, Lys-10, and Lys-16), which mediate electrostatic interactions with anionic membranes, primarily accelerate the membrane association. In contrast, an aliphatic side chain of the C2 domain of cytosolic PLA(2) (Val-97), which penetrates into the hydrophobic core of the membrane and forms hydrophobic interactions, mainly slows the dissociation of membrane-bound protein. Aromatic residues of human group V PLA(2) (Trp-31) and N. n. atra PLA(2) (Trp-61, Phe-64, and Tyr-110) contribute to both membrane association and dissociation steps, and the relative contribution to these processes depends on the chemical nature and the orientation of the side chains as well as their location on the interfacial binding surface. On the basis of these results, a general model is proposed for the interfacial binding of peripheral proteins, in which electrostatic interactions by ionic and aromatic residues initially bring the protein to the membrane surface and the subsequent membrane penetration and hydrophobic interactions by aliphatic and aromatic residues stabilize the membrane-protein complexes, thereby elongating the membrane residence time of protein.

The FYVE domain mediates the recruitment of proteins involved in membrane trafficking and cell signaling to phosphatidylinositol 3-phosphate (PtdIns(3)P)-containing membranes. To elucidate the mechanism by which the FYVE domain interacts with PtdIns(3)P-containing membranes, we measured the membrane binding of the FYVE domains of yeast Vps27p and Drosophila hepatocyte growth factor-regulated tyrosine kinase substrate and their mutants by surface plasmon resonance and monolayer penetration analyses. These measurements as well as electrostatic potential calculation show that PtdIns(3)P specifically induces the membrane penetration of the FYVE domains and increases their membrane residence time by decreasing the positive charge surrounding the hydrophobic tip of the domain and causing local conformational changes. Mutations of hydrophobic residues located close to the PtdIns(3)P-binding pocket or an Arg residue directly involved in PtdIns(3)P binding abrogated the penetration of the FYVE domains into the monolayer, the packing density of which is comparable with that of biological membranes and large unilamellar vesicles. Based on these results, we propose a mechanism of the membrane binding of the FYVE domain in which the domain first binds to the PtdIns(3)P-containing membrane by specific PtdIns(3)P binding and nonspecific electrostatic interactions, which is then followed by the PtdIns(3)P-induced partial membrane penetration of the domain.FYVE domains are small (70 -80 amino acids) cysteine-rich domains that bind two zinc ions (1, 2). They are named for the first letters of the first four proteins in which they were identified: Fablp, YOTB, Vac1p, and early endosomal antigen 1 (EEA1).1 A functional role for the FYVE domain was first recognized when it was found to facilitate the localization of EEA1 to endosomes (3). Thereafter, it has been demonstrated that the FYVE domain specifically binds phosphatidylinositol 3-phosphate (PtdIns(3)P) in vitro (4, 5). PtdIns(3)P is constitutively present in eukaryotic cells, and the majority of PtdIns(3)P in mammalian cells is produced by class III phosphoinositide 3-kinase (6). PtdIns(3)P is thought to be involved in vesicle trafficking as mutations and inhibition of phosphoinositide 3-kinase have caused mis-sorting of vacuolar proteins, changes in vacuole morphology, and defects in the endocytic pathway (7). As expected from proposed roles of PtdIns(3)P, it has been found in specific subcellular locales, including the cytoplasmic face of early endosomes and internal vesicles of multivesicular bodies (6). Consistent with its in vitro PtdIns(3)P specificity, the FYVE domain has been also shown to be localized to these membrane sites in vivo (8, 9). Recent structural analyses of several FYVE domains (10 -13), a crystallographic structure analysis of EEA1-FYVE in particular (13), have elucidated the mechanism by which FYVE domains specifically recognize the PtdIns(3)P molecule. However, less is known about the mechanism by which FYVE domains interact with and are targeted to P...

The regulatory domain of conventional protein kinase C (PKC) contains two membrane-targeting modules, the C2 domain that is responsible for Ca 2؉ -dependent membrane binding of protein, and the C1 domain composed of two cysteine-rich zinc fingers (C1a and C1b) that bind diacylglycerols and phorbol esters. To understand the individual roles and the interplay of the C1 and C2 domains in the membrane binding and activation of PKC, we functionally expressed isolated C1 and C2 domains of PKC-␣ and measured their vesicle binding and monolayer penetration. Results indicate that the C2 domain of PKC-␣ is responsible for the initial Ca 2؉ -and phosphatidylserine-dependent electrostatic membrane binding of PKC-␣, whereas the C1 domain is involved in subsequent membrane penetration and diacylglycerol binding, which eventually lead to enzyme activation. To determine the roles of individual zinc fingers in the C1 domain, we also mutated hydrophobic residues in the C1a (Trp 58 and Protein kinases C (PKCs)1 are a family of serine/threonine kinases that transduce the myriad of signals activating cellular functions and proliferation (1, 2). More than 10 members of the PKC family have been identified by molecular cloning. All PKCs contain an amino-terminal regulatory domain and a carboxyl-terminal catalytic domain. Based on structural differences in the regulatory domain, PKCs are generally classified into three groups; conventional PKC (␣, ␤I, ␤II, and ␥ subtypes), novel PKC (␦, ⑀, , and subtypes), and atypical PKC ( and subtypes). The regulatory domain of conventional PKCs is composed of two conserved membrane-targeting modules, C1 and C2 domains, as well as a pseudosubstrate region and variable regions. Conventional PKCs are activated by the Ca 2ϩ -dependent translocation of proteins to the membrane containing phosphatidylserine (PS) and diacylglycerol (DAG). Structural (3) and mutational (4, 5) studies have shown that the C2 domain of conventional PKC is responsible for the Ca 2ϩ -dependent translocation of the protein to membranes. It has also been shown that the C1 domain, which is composed of a tandem repeat of cysteine-rich zinc-finger domains (C1a and C1b), is involved in binding of PKC to DAG and its structural analogs, phorbol esters (6 -8). However, the temporal and spatial sequences of membrane targeting and activation of PKC have not been fully elucidated. Furthermore, the interplay of the C1 and C2 domains in these complex processes is poorly understood. Finally, the roles of individual zinc finger domains in the DAGdependent membrane binding and activation of PKC are not well defined. To address these questions, we functionally expressed the isolated C1 and C2 domains of PKC-␣ and measured their vesicle binding and monolayer penetration. We also mutated C1a and C1b domain residues of the native PKC-␣ and measured the effects of mutations on vesicle binding, enzyme activity, and monolayer penetration. Results indicate that the C2 domain of PKC-␣ is responsible for its initial Ca 2ϩ -and PS-dependent membrane bind...