Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is an antagonist of class I phosphatidylinositol 3—kinase (PI3K) signaling. In response to extracellular stimuli (e.g., insulin, growth factors, chemokines), the catalytic subunit of PI3K (p110) is recruited to receptor tyrosine kinases (RTKs) or G protein—coupled receptors (GPCRs) at the membrane through its regulatory subunit (p85 or p101), where it phosphorylates phosphatidylinositol-4,5 bisphosphate (PIP₂) to generate phosphatidylinositol-3,4,5 trisphosphate (PIP₃). PTEN is a lipid phosphatase that antagonizes the action of PI3K by dephosphorylating PIP₃ at position D3 to generate PIP₂.

p110α protein structure and mutation distribution. (a) p110α is characterized by five functional domains: p85-regulatory subunit—binding domain (p85-BD), Ras-binding domain (Ras-BD), C2 domain, helical domain, and kinase catalytic domain. The percentage of mutations identified in each domain is shown at bottom. (b) Distribution of cancer-specific mutations in PIK3CA and their relative frequency of occurrence in the functional domains. The three hot spots for mutations (E542, E545, H1047) are depicted. Values are taken from the Catalogue of Somatic Mutations in Cancer (COSMIC) database (http://www.sanger.ac.uk/genetics/CGP/cosmic) and include single substitutions and complex mutations. Amino acid numbers are listed along the x axis, with the corresponding PIK3CA exon structure encoding p110α shown in blue boxes below. The numbers of mutations are listed along the y axis. Abbreviations: ATG, start codon; TGA, stop codon.

PTEN (phosphatase and tensin homolog deleted on chromosome 10) protein structure and mutation distribution. (a) PTEN is composed of 403 amino acids and is characterized by five functional domains: a phosphatidylinositol-4,5-bisphosphate (PIP₂)-binding domain (PBD), a phosphatase domain highlighted by cancer-specific mutations in its catalytic core, a C2 domain with a putative ubiquitination residue associated with cancer, two PEST (proline, glutamic acid, serine, threonine) domains for degradation, and a PDZinteraction motif for protein-protein interactions. Also depicted are an N-terminal nuclear localization signal-like region, a C-terminal nuclear exclusion signal, and several charged residues and phosphorylation sites important for subcellular localization and stability. Several mechanisms regulating the activity of PTEN are shown. (b) Distribution of cancer-specific mutations found in PTEN and their percentage of occurrence in the functional domains. Values are taken from the Catalogue of Somatic Mutations in Cancer (COSMIC) database (http://www.sanger.ac.uk/genetics/CGP/cosmic") and include single substitutions and complex mutations. Amino acid numbers are listed along the x axis with the corresponding PTEN exon structure shown in blue boxes below, and the numbers of mutations are listed along the y axis. Abbreviations: ATG, start codon; NLS, nuclear localization signal; PDZ, postsynaptic density protein—Drosophila disc large tumor suppressor—zonula occludens 1 protein; TGA, stop codon.

Pten (phosphatase and tensin homolog deleted on chromosome 10) protein localizes to multiple subcellular compartments. Pten conditional knockout brains contain Pten-positive and Pten-negative neurons, providing internal controls for immunohistochemical detection of Pten. In mature neurons of the cerebral cortex, Pten is present in the nucleus, the soma, and the neuronal projections (yellow arrowheads). Pten-deficient neurons show global hypertrophy, and the absence of Pten is seen clearly in all cell compartments (red arrowheads). Immunohistochemical detection of Pten was performed with anti-Pten antibody from cell signaling (#9559), peroxidase-labeled secondary antibody, diaminobenzidine (DAB) substrate, and hematoxylin counterstain.

The phosphatidylinositol 3—kinase (PI3K) signaling pathway. Activated receptor tyrosine kinases (RTKs) recruit and activate PI3K, leading to increased phosphatidylinositol-3,4,5-trisphosphate (PIP₃) levels. PIP₃ recruits many proteins to the membrane by binding to their pleckstrin homology (PH) domains, including the serine/threonine kinases AKT, 3-phosphoinositide-dependent kinase (PDK1), and the phosphatase PH domain and leucine rich repeat protein phosphatase (PHLPP). Membrane-bound AKT is rendered fully active through its phosphorylation by PDK1 and the rapamycin-insensitive mammalian target of rapamycin (mTOR) complex (mTORC2), and it is inactivated when dephosphorylated by PHLPP. Activated AKT may phosphorylate a range of substrates, thereby activating or inhibiting these targets and resulting in cellular growth, survival, and proliferation through various mechanisms. PI3K can also regulate downstream targets, such as RAC1/CDC42, in an AKT-independent manner. Activation of the rapamycin-sensitive mTOR complex (mTORC1) is inhibited by the tuberous sclerosis complex (TSC1 and TSC2) 1 and 2, which can be regulated by AKT as well as through PI3K-independent signaling from LKB1 and AMP-activated protein kinase (AMPK). Abbreviations: GSK, glycogen synthase kinase; NF-κB, nuclear factor—κ B; PIP₂, phosphatidylinositol-4,5 bisphosphate; RAC1, Ras-related C3 botulinum toxin substrate 1.

Mechanistic model of enhanced activation induced by the E545K mutation in p110α. Based on the crystal structure of p110α, the following model has been proposed. The wild-type catalytic p110α subunit is stabilized by binding to the p85α-regulatory subunit through its p85 binding domain (BD). In addition, its activity is maintained at a low state through interaction of its negatively charged helical domain with the positively charged N-terminal inter-Src homology 2 (nSH2) domain of p85α. Receptor tyrosine kinase (RTK) activation and its subsequent phosphorylation recruits the p85α/p110α complex to the membrane, causing conformational changes that relieve the charge-inhibitory interactions of p85α on p110α and lead to high transitional p110α activity. In the presence of the E545K mutation, the negative charge is converted to a positive charge, resulting in the abrogation of the p85α/p110α charge-inhibitory interactions, sustained p110α activity at the membrane, and ultimately delayed dissociation of the p85α subunit from the phospho-RTK. Abbreviations: cSH2, C-terminal inter-Src homology 2; iSH2, inter-Src homology 2; PI3K, phosphatidylinositol 3—kinase; PIP₂, phosphatidylinositol-4,5-bisphosphate; PIP₃, phosphatidylinositol-3,4,5-trisphosphate. Adapted from Lee et al. (31a).

Regulation of PTEN (phosphatase and tensin homolog deleted on chromosome 10) protein stability and localization. PTEN can be regulated by several mechanisms, including phosphorylation and ubiquitination, that govern its stability and activity through its subcellular localization. When phosphorylated at the C terminus, PTEN adopts a closed conformation that increases its stability but decreases its activity. Conversely, dephosphorylation of PTEN leads to its recruitment to the membrane, where it may interact with PDZ (postsynaptic density protein—Drosophila disc large tumor suppressor—zonula occludens 1 protein) domain-containing proteins through its PDZ interaction motif. Membrane-associated PTEN is more active but less stable. In addition, PTEN turnover can be regulated by NEDD4–1-mediated ubiquitination. Polyubiquitinated PTEN remains in the cytoplasm and is targeted for degradation by the proteasome, whereas monoubiquitination of PTEN appears to regulate import of PTEN into the nucleus, where its function remains unclear. Abbreviations: PIP₂, phosphatidylinositol-4,5-bisphosphate; PIP₃, phosphatidylinositol-3,4,5 trisphosphate.

Feedback inhibition of the phosphatidylinositol 3—kinase (PI3K) pathway. Activated AKT regulates cellular growth through mammalian target of rapamycin (mTOR), a key player in protein synthesis and translation. mTOR forms part of two distinct complexes known as mTORC1 (which contains mTOR, Raptor, mLST8, and PRAS40) and mTORC2 (which contains mTOR, Rictor, mLST8, and mSIN1). mTORC1 is sensitive to rapamycin and controls protein synthesis and translation, at least in part, through p70S6K and eukaryotic translation initiation factor 4E—binding protein 1 (4E-BP1). AKT phosphorylates and inhibits tuberous sclerosis complex 2 (TSC2), resulting in increased mTORC1 activity. AKT also phosphorylates PRAS40, thus relieving the PRAS40 inhibitory effect on mTOR and the mTORC1 complex. mTORC2 and 3-phosphoinositide-dependent kinase (PDK1) phosphorylate AKT on Ser473 and Thr308, respectively, rendering it fully active. mTORC1-activated p70S6K can phosphorylate insulin receptor substrate 1 (IRS1), resulting in inhibition of PI3K activity. In addition, PDK1 phosphorylates and activates p70S6K and p90S6K. The latter has been shown to inhibit TSC2 activity through direct phosphorylation. Conversely, LKB1-activated AMP-activated protein kinase (AMPK) and glycogen synthase kinase 3 (GSK3) activate the TSC1/TSC2 complex through direct phosphorylation of TSC2. Thus, signals through PI3K as well as through LKB1 and AMPK converge on mTORC1. Inhibition of mTORC1 can lead to increased insulin receptor—mediated signaling, and inhibition of PDK1 may lead to activation of mTORC1 and may, paradoxically, promote tumor growth.