Spatial compartmentalization of signaling pathway components generally defines the specificity and enhances the efficiency of signal transduction. The phosphatidylinositol 3-kinase (PI3K)/Akt pathway is known to be compartmentalized within plasma membrane microdomains; however, the underlying mechanisms and functional impact of this compartmentalization are not well understood. Here, we show that phosphoinositide-dependent kinase 1 is activated in membrane rafts in response to growth factors, whereas the negative regulator of the pathway, phosphatase and tensin homolog deleted on chromosome 10 (PTEN), is primarily localized in nonraft regions. Alteration of this compartmentalization, either by genetic targeting or ceramide-induced recruitment of PTEN to rafts, abolishes the activity of the entire pathway. These findings reveal critical steps in raft-mediated PI3K/Akt activation and demonstrate the essential role of membrane microdomain compartmentalization in enabling PI3K/Akt signaling. They further suggest that dysregulation of this compartmentalization may underlie pathological complications such as insulin resistance.

Wnts are secreted palmitoylated glycoproteins that are important in embryonic development and human cancers. Here we report a method for imaging the palmitoylated form of Wnt proteins with subcellular resolution using clickable bioorthogonal fatty acids and in situ proximity ligation. Palmitoylated Wnt3a is visualized throughout the secretory pathway and trafficks to multivesicular bodies that act as export sites in secretory cells. We establish that glycosylation is not required for Wnt3a palmitoylation, which is necessary but not sufficient for Wnt3a secretion. Wnt3a is palmitoylated by fatty acids 13-16 carbons in length at Ser209 but not at Cys77, consistent with a slow turnover rate. We find that porcupine (PORCN) itself is palmitoylated, demonstrating what is to our knowledge the first example of palmitoylation of an MBOAT protein, and this modification partially regulates Wnt palmitoylation and signaling. Our data reveal the role of O-palmitoylation in Wnt signaling and suggest another layer of cellular control over PORCN function and Wnt secretion.

As a central kinase in the phosphatidylinositol 3-kinase pathway, Akt has been the subject of extensive research; yet, spatiotemporal regulation of Akt in different membrane microdomains remains largely unknown. To examine dynamic Akt activity in membrane microdomains in living cells, we developed a specific and sensitive fluorescence resonance energy transfer-based Akt activity reporter, AktAR, through systematic testing of different substrates and fluorescent proteins. Targeted AktAR reported higher Akt activity with faster activation kinetics within lipid rafts compared with nonraft regions of plasma membrane. Disruption of rafts attenuated platelet-derived growth factor (PDGF)-stimulated Akt activity in rafts without affecting that in nonraft regions. However, in insulin-like growth factor-1 (IGF)-1 stimulation, Akt signaling in nonraft regions is dependent on that in raft regions. As a result, cholesterol depletion diminishes Akt activity in both regions. Thus, Akt activities are differentially regulated in different membrane microdomains, and the overall activity of this oncogenic pathway is dependent on raft function. Given the increased abundance of lipid rafts in some cancer cells, the distinct Akt-activating characteristics of PDGF and IGF-1, in terms of both effectiveness and raft dependence, demonstrate the capabilities of different growth factor signaling pathways to transduce differential oncogenic signals across plasma membrane. INTRODUCTIONHuman serine-threonine kinase Akt (Bellacosa et al., 1991) was first identified as an oncogene in 1991, followed by discovery and characterization of many important upstream and downstream regulatory components (Brazil and Hemmings, 2001). There are three mammalian Akt isoforms, all of which share similar domain structures that include three functional domains, namely, an N-terminal pleckstrin homology (PH) domain, a kinase domain, and a C-terminal regulatory hydrophobic motif (Frech et al., 1997;Vivanco and Sawyers, 2002). As a key cellular regulator that transduces various signals that turn on phosphatidylinositol 3-kinase (PI3K), the activity of Akt is dynamically regulated. On growth factor stimulation, active PI3K catalyzes the conversion of phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P 2 ] into the cell membrane-bound second messenger phosphatidylinositol (3,4,5)-triphosphate [PI(3,4,5)P 3 ], which recruits Akt to plasma membrane, where 3-phosphoinositide-dependent protein kinase 1 phosphorylates the T-loop of Akt at a threonine residue (Thr 308/309) (Alessi et al., 1997). Another phosphorylation event mediated by mammalian target of rapamycin-Rictor complex occurs at a serine residue located in the C-terminal hydrophobic motif of Akt (Ser 473/474) (Sarbassov et al., 2005), leading to full activation of Akt. A tumor suppressor, phosphatase and tensin homologue deleted on chromosome 10 (PTEN), dephosphorylates PI(3, 4,5)P 3 to PI(4,5)P 2 , suppressing Akt activation through reduction of the second messenger (Li and Ross, 2007). Two phosphatases, protei...

A high-resolution map of human phosphorylation networks was constructed by integrating experimentally determined kinase-substrate relationships with other resources, such as in vivo phosphorylation sites.

Many protein kinases are key nodal signaling molecules that regulate a wide range of cellular functions. These functions may require complex spatiotemporal regulation of kinase activities. Here, we show that Protein Kinase A (PKA), Ca2+ and cAMP oscillate in sync in insulin-secreting MIN6 β cells, forming a highly integrated oscillatory circuit. We found that PKA activity was essential for this oscillatory circuit, and was capable of not only initiating the signaling oscillations but also modulating their frequency, thereby diversifying the spatiotemporal control of downstream signaling. Our findings suggest that exquisite temporal control of kinase activity, mediated via signaling circuits resulting from cross-regulation of signaling pathways, can encode diverse inputs into temporal parameters such as oscillation frequency, which in turn contributes to proper regulation of complex cellular functions in a context-dependent manner.

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Protein palmitoylation plays diverse roles in regulating the trafficking, stability, and activity of cellular proteins. The advent of click chemistry has propelled the field of protein palmitoylation forward by providing specific, sensitive, rapid, and easy-to-handle methods for studying protein palmitoylation. This year marks the 10th anniversary since the first click chemistry-based fatty acid probes for detecting protein lipid modifications were reported. The goal of this review is to highlight key biological advancements in the field of protein palmitoylation during the past 10 years. In particular, we discuss the impact of click chemistry on enabling protein palmitoylation proteomics methods, uncovering novel lipid modifications on proteins and elucidating their functions, as well as the development of non-radioactive biochemical and enzymatic assays. In addition, this review provides context for building and exploring new research avenues in protein palmitoylation through the use of clickable fatty acid probes.

Hedgehog protein undergoes post-translational palmitoylation, which is critical for its signaling activity during embryonic development and in adult tissues. Due to a lack of suitable imaging methods, the trafficking route of palmitoylated Hedgehog has remained unclear in secretory cells. Here, we report a novel method for imaging the subcellular distribution of palmitoylated forms of cellular proteins with high resolution. The method utilizes clickable chemical reporters to label the entire palmitoylated proteome, followed by proximity ligation on antibodies to the click-conjugated dye and the protein of interest to reveal the spatial localization of specific palmitoylated proteins, as exemplified by sonic Hedgehog, tubulin, and Ras. Palmitoylated sonic Hedgehog is found in the endoplasmic reticulum, the Golgi apparatus, and at the plasma membrane but not the endosomal system in Hedgehog-secreting cells. Palmitoylated tubulin is found along microtubule tracks and also partially associated with the plasma membrane, while palmitoylated H-Ras is visualized at various cellular locations including the plasma membrane, Golgi apparatus and endoplasmic reticulum. Our method is broadly applicable to imaging the palmitoylation of cellular proteins as well as other protein post-translational modifications that are detectable by clickable chemical reporters.