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Selenoprotein P (SeP) is one of the 25 human selenocysteine (Sec)-containing proteins, and is generally thought to function as a plasma carrier of the trace element selenium in the body. Recent studies, however, indicate unsuspected pivotal roles of SeP in human diseases, particularly in type 2 diabetes mellitus (T2DM) and pulmonary arterial hypertension (PAH). In this review, we will summarize the characteristics of SeP and recent advances in the field, especially focusing on the emerging roles of SeP in pathophysiological conditions. We will also discuss potential medical/pharmaceutical applications targeting SeP.

Due to the involvement of SHP2 (SH2 domain‐containing protein‐tyrosine phosphatase) in human disease, including Noonan syndrome and cancer, several inhibitors targeting SHP2 have been developed. Here, we report that the commonly used SHP2 inhibitor NSC‐87877 does not exhibit robust inhibitory effects on growth factor‐dependent MAPK (mitogen‐activated protein kinase) pathway activation and that the recently developed active site‐targeting SHP2 inhibitors IIB‐08, 11a‐1, and GS‐493 show off‐target effects on ligand‐evoked activation/trans‐phosphorylation of the PDGFRβ (platelet‐derived growth factor receptor β). GS‐493 also inhibits purified human PDGFRβ and SRC in vitro, whereas PDGFRβ inhibition by IIB‐08 and 11a‐1 occurs only in the cellular context. Our results argue for extreme caution in inferring specific functions for SHP2 based on studies using these inhibitors.

The physiological importance of reactive sulfur species (RSS) such as cysteine hydropersulfide (CysSSH) has been increasingly recognized in recent years. We have established a reactive sulfur metabolomics analysis by using RSS metabolic profiling, which revealed appreciable amounts of RSS generated endogenously and ubiquitously in both prokaryotic and eukaryotic organisms. The chemical nature of these polysulfides is not fully understood, however, because of their reactive or complicated redox-active properties. In our study here, we determined that tyrosine and a hydroxyphenyl-containing derivative, β-(4-hydroxyphenyl)ethyl iodoacetamide (HPE-IAM), had potent stabilizing effects on diverse polysulfide residues formed in CysSSH-related low-molecular-weight species, e.g., glutathione polysulfides (oxidized glutathione trisulfide and oxidized glutathione tetrasulfide). The protective effect against degradation was likely caused by the inhibitory activity of hydroxyphenyl residues of tyrosine and HPE-IAM against alkaline hydrolysis of polysulfides. This hydrolysis occurred via heterolytic scission triggered by the hydroxyl anion acting on polysulfides that are cleaved into thiolates and sulfenic acids, with the hydrolysis being enhanced by alkylating reagents (e.g. IAM) and dimedone. Moreover, tyrosine prevented electrophilic degradation occurring in alkaline pH. The polysulfide stabilization induced by tyrosine or the hydroxyphenyl moiety of HPE-IAM will greatly improve our understanding of the chemical properties of polysulfides and may benefit the sulfur metabolomics analysis if it can be applied successfully to any kind of biological samples, including clinical specimens., Graphical abstract fx1, Highlights • Polysulfides undergo hydrolysis under alkaline pH conditions. • Alkylating reagents and dimedone enhance polysulfide decomposition. • Tyr and hydroxyphenyl derivatives inhibit alkaline-induced polysulfide hydrolysis. • Tyr protects polysulfides from electrophile- and dimedone-enhanced hydrolysis.

Due to the involvement of SHP2 (SH2 domain-containing protein-tyrosine phosphatase) in human disease, including Noonan syndrome and cancer, several inhibitors targeting SHP2 have been developed. Here, we report that the commonly used SHP2 inhibitor NSC-87877 does not exhibit robust inhibitory effects on growth factor-dependent MAPK (mitogen-activated protein kinase) pathway activation and that the recently developed active site-targeting SHP2 inhibitors IIB-08, 11a-1, and GS-493 show off-target effects on ligand-evoked activation/trans-phosphorylation of the PDGFRβ (platelet-derived growth factor receptor β). GS-493 also inhibits purified human PDGFRβ and SRC

Due to the involvement of SHP2 (SH2 domain-containing protein tyrosine phosphatase) in human disease, including Noonan syndrome and cancer, several inhibitors targeting SHP2 have been developed. Here, we report that the commonly used SHP2 inhibitor NSC-78788 does not exhibit robust inhibitory effects on growth factor-dependent MAPK (mitogen-activated protein kinase) pathway activation, and that the recently developed active site-targeting SHP2 inhibitors IIB-08, 11a-1, and GS-493 show off-target effects on ligand-evoked activation/trans-phosphorylation of the PDGFRβ (platelet-derived growth factor receptor β). GS-493 also inhibits purified human PDGFRβ and SRC in vitro, whereas PDGFRβ inhibition by IIB-08 and 11a-1 occurs only in the cellular context. Our results argue for extreme caution in inferring specific functions for SHP2 based on studies using these inhibitors.

Reactive oxygen species are produced transiently in response to cell stimuli, and function as second messengers that oxidize target proteins. Protein-tyrosine phosphatases are important reactive oxygen species targets, whose oxidation results in rapid, reversible, catalytic inactivation. Despite increasing evidence for the importance of protein-tyrosine phosphatase oxidation in signal transduction, the cell biological details of reactive oxygen species-catalyzed protein-tyrosine phosphatase inactivation have remained largely unclear, due to our inability to visualize protein-tyrosine phosphatase oxidation in cells. By combining proximity ligation assay with chemical labeling of cysteine residues in the sulfenic acid state, we visualize oxidized Src homology 2 domain-containing protein-tyrosine phosphatase 2 (SHP2). We find that platelet-derived growth factor evokes transient oxidation on or close to RAB5+/ early endosome antigen 1− endosomes. SHP2 oxidation requires NADPH oxidases (NOXs), and oxidized SHP2 co-localizes with platelet-derived growth factor receptor and NOX1/4. Our data demonstrate spatially and temporally limited protein oxidation within cells, and suggest that platelet-derived growth factor-dependent “redoxosomes,” contribute to proper signal transduction., Protein-tyrosine phosphatases (PTPs) are thought to be major targets of receptor-activated reactive oxygen species (ROS). Here the authors describe a method that allows the localized visualization of oxidized intermediates of PTPs inside cells during signaling, and provide support for the “redoxosome” model.

SummarySHP2 is a ubiquitously expressed protein tyrosine phosphatase, deregulation of which is associated with malignant neoplasms and developmental disorders. SHP2 is required for full activation of RAS-Erk signaling in the cytoplasm and is also present in the nucleus, where it promotes Wnt target gene activation through dephosphorylation of parafibromin. SHP2 is distributed both to the cytoplasm and nucleus at low cell density but is excluded from the nucleus at high cell density. Here, we show that SHP2 physically interacts with transcriptional coactivators YAP and TAZ, targets of the cell-density-sensing Hippo signal. Through the interaction, nonphosphorylated YAP/TAZ promote nuclear translocalization of SHP2, which in turn stimulates TCF/LEF- and TEAD-regulated genes via parafibromin dephosphorylation. Conversely, YAP/TAZ phosphorylated by Hippo signaling sequester SHP2 in the cytoplasm, thereby preventing nuclear accumulation of SHP2. Hence, YAP/TAZ serve as a rheostat for nuclear SHP2 function, which is switched off by the Hippo signal.

The non-receptor protein tyrosine phosphatase SHP2, encoded by PTPN11, has an important role in signal transduction downstream of growth factor receptor signalling and was the first reported oncogenic tyrosine phosphatase. Activating mutations of SHP2 have been associated with developmental pathologies such as Noonan syndrome and are found in multiple cancer types, including leukaemia, lung and breast cancer and neuroblastoma. SHP2 is ubiquitously expressed and regulates cell survival and proliferation primarily through activation of the RAS–ERK signalling pathway. It is also a key mediator of the programmed cell death 1 (PD-1) and B- and T-lymphocyte attenuator (BTLA) immune checkpoint pathways. Reduction of SHP2 activity suppresses tumour cell growth and is a potential target of cancer therapy. Here we report the discovery of a highly potent (IC50 = 0.071 μM), selective and orally bioavailable small-molecule SHP2 inhibitor, SHP099, that stabilizes SHP2 in an auto-inhibited conformation. SHP099 concurrently binds to the interface of the N-terminal SH2, C-terminal SH2, and protein tyrosine phosphatase domains, thus inhibiting SHP2 activity through an allosteric mechanism. SHP099 suppresses RAS–ERK signalling to inhibit the proliferation of receptor-tyrosine-kinase-driven human cancer cells in vitro and is efficacious in mouse tumour xenograft models. Together, these data demonstrate that pharmacological inhibition of SHP2 is a valid therapeutic approach for the treatment of cancers.

Protein palmitoylation is the most common posttranslational lipid modification; its reversibility mediates protein shuttling between intracellular compartments. A large family of DHHC (Asp-His-His-Cys) proteins has emerged as protein palmitoyl acyltransferases (PATs). However, mechanisms that regulate these PATs in a physiological context remain unknown. In this study, we efficiently monitored the dynamic palmitate cycling on synaptic scaffold PSD-95. We found that blocking synaptic activity rapidly induces PSD-95 palmitoylation and mediates synaptic clustering of PSD-95 and associated AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)-type glutamate receptors. A dendritically localized DHHC2 but not the Golgi-resident DHHC3 mediates this activity-sensitive palmitoylation. Upon activity blockade, DHHC2 translocates to the postsynaptic density to transduce this effect. These data demonstrate that individual DHHC members are differentially regulated and that dynamic recruitment of protein palmitoylation machinery enables compartmentalized regulation of protein trafficking in response to extracellular signals.

Protein S-palmitoylation, the most common lipid modification with the 16-carbon fatty acid palmitate, provides an important mechanism for regulating protein trafficking and function. The unique reversibility of protein palmitoylation allows proteins to rapidly shuttle between intracellular membrane compartments. Importantly, this palmitate cycling can be regulated by some physiological stimuli, contributing to cellular homeostasis and plasticity. Although the enzyme responsible for protein palmitoylation had been long elusive, DHHC family proteins, conserved from plants to mammals, have recently emerged as palmitoyl acyl transferases. Integrated approaches including advanced proteomics, live-cell imaging, and molecular genetics are beginning to clarify the molecular machinery for palmitoylation reaction in diverse aspects of cellular functions.

The heterotrimeric G protein alpha subunit (Galpha) is targeted to the cytoplasmic face of the plasma membrane through reversible lipid palmitoylation and relays signals from G-protein-coupled receptors (GPCRs) to its effectors. By screening 23 DHHC motif (Asp-His-His-Cys) palmitoyl acyl-transferases, we identified DHHC3 and DHHC7 as Galpha palmitoylating enzymes. DHHC3 and DHHC7 robustly palmitoylated Galpha(q), Galpha(s), and Galpha(i2) in HEK293T cells. Knockdown of DHHC3 and DHHC7 decreased Galpha(q/11) palmitoylation and relocalized it from the plasma membrane into the cytoplasm. Photoconversion analysis revealed that Galpha(q) rapidly shuttles between the plasma membrane and the Golgi apparatus, where DHHC3 specifically localizes. Fluorescence recovery after photobleaching studies showed that DHHC3 and DHHC7 are necessary for this continuous Galpha(q) shuttling. Furthermore, DHHC3 and DHHC7 knockdown blocked the alpha(1A)-adrenergic receptor/Galpha(q/11)-mediated signaling pathway. Together, our findings revealed that DHHC3 and DHHC7 regulate GPCR-mediated signal transduction by controlling Galpha localization to the plasma membrane.

Posttranslational modification provides proteins with additional function and regulatory control beyond genomic information, allowing cells to maintain homeostasis and respond to extracellular signals. Protein palmitoylation, the common posttranslational modification with the lipid palmitate, plays a pivotal role in protein trafficking and function. Palmitoylation is unique in that it is reversible and dynamically regulated by specific extracellular signals. The reversible nature of protein palmitoylation enables proteins to shuttle between intracellular compartments upon extracellular signals. However, the molecular mechanisms of protein palmitoylation have long been elusive, mostly because the enzymes responsible for protein palmitoylation were unknown. Recently, genetically conserved DHHC family proteins have emerged as palmitoyl-acyl transferases. With the identification of specific enzymes for palmitoylated proteins, including H-Ras, PSD-95, and eNOS, the specificity and regulatory mechanism of DHHC enzymes are beginning to be clarified.

Background & Aims: Helicobacter pylori CagA-positive strain is associated with gastric adenocarcinoma. CagA is delivered into gastric epithelial cells, where it undergoes tyrosine phosphorylation at the EPIYA sites by Src family kinases (SFKs). Owing to homologous recombination within the 3′-region of the cagA gene, 4 distinct EPIYA sites, each of which is defined by surrounding sequences, are variably assembled in both number and order among CagA proteins from different clinical H pylori isolates. Tyrosine-phosphorylated CagA specifically binds and deregulates SHP-2 via the Western CagA-specific EPIYA-C or East Asian CagA-specific EPIYA-D site, and C-terminal Src kinase (Csk) via the EPIYA-A or EPIYA-B site. Here we investigated the influence of EPIYA-repeat polymorphism on the CagA activity. Methods: A series of EPIYA-repeat variants of CagA were expressed in AGS gastric epithelial cells and the ability of individual CagA to bind SHP-2 or Csk was determined by the sequential immunoprecipitation and immunoblotting method. Results: CagA proteins carrying multiple EPIYA-C or EPIYA-D sites bound and deregulated SHP-2 more strongly than those having a single EPIYA-C or EPIYA-D. Furthermore, the ability of CagA to bind Csk was correlated with the number of EPIYA-A and EPIYA-B sites. Because Csk inhibits SFK, CagA with greater Csk-binding activity more strongly inhibited Src-dependent CagA phosphorylation and more effectively attenuated induction of cell elongation caused by CagA–SHP-2 interaction. Conclusions: EPIYA-repeat polymorphism of CagA greatly influences the magnitude and duration of phosphorylation-dependent CagA activity, which may determine the potential of individual CagA as a bacterial virulence factor that directs gastric carcinogenesis.

RNA interference (RNAi) is a newly described biological phenomenon mediated by small interfering RNA (siRNA) that targets mRNA for degradation by cellular enzymes and has become a powerful method for studying gene functions in mammalian systems. The development of systems for inducing siRNA expression should enable examination of acute loss-of-function phenotypes in a cell of interest without the need to consider lethality or epigenetic adaptation of cells. We describe in this report an inducible siRNA expression system made by combined utilization of the RNA polymerase III-dependent promoter H1 and the bacterial lac repressor. Using this system, we established AGS gastric epithelial cells in which expression of SHP-2, a cellular tyrosine phosphatase known to specifically bind the Helicobacter pylori virulence factor CagA, is conditionally and reversibly silenced by the lactose analog isopropyl-1-thio-beta-D-galactopyranoside (IPTG). Upon expression in AGS cells, CagA provoked a morphological transformation, termed the hummingbird phenotype, which is associated with CagA virulence. This morphogenetic activity of CagA was totally abolished when SHP-2 expression was silenced by inducible siRNA expression in AGS cells. Our results indicate that SHP-2 is a critical downstream effector of H. pylori CagA. The conditional gene silencing system described here should become a powerful tool for investigating the roles of cancer-related genes through a reversed genetic approach.

The CagA protein of Helicobacter pylori, which is injected from the bacteria into bacteria-attached gastric epithelial cells, is associated with gastric carcinoma. CagA is tyrosine-phosphorylated by Src family kinases, binds the SH2 domain-containing SHP-2 phosphatase in a tyrosine phosphorylation-dependent manner, and deregulates its enzymatic activity. We established AGS human gastric epithelial cells that inducibly express wild-type or a phosphorylation-resistant CagA, in which tyrosine residues constituting the EPIYA motifs were substituted with alanines. Upon induction, wild-type CagA, but not the mutant CagA, elicited strong elongation of cell shape, termed the "hummingbird" phenotype. Time-lapse video microscopic analysis revealed that the CagA-expressing cells exhibited a marked increase in cell motility with successive rounds of elongation-contraction processes. Inhibition of CagA phosphorylation by an Src kinase inhibitor, PP2, or knockdown of SHP-2 expression by small interference RNA (siRNA) abolished the CagA-mediated hummingbird phenotype. The morphogenetic activity of CagA also required Erk MAPK but was independent of Ras or Grb2. In AGS cells, CagA prolonged duration of Erk activation in response to serum stimulation. Conversely, inhibition of SHP-2 expression by siRNA abolished the sustained Erk activation. Thus, SHP-2 acts as a positive regulator of Erk activity in AGS cells. These results indicate that SHP-2 is involved in the Ras-independent modification of Erk signals that is necessary for the morphogenetic activity of CagA. Our work therefore suggests a key role of SHP-2 in the pathological activity of H. pylori virulence factor CagA.

Helicobacter pylori is a causative agent of gastritis and peptic ulcer. cagA + H. pylori strains are more virulent than cagA − strains and are associated with gastric carcinoma. The cagA gene product, CagA, is injected by the bacterium into gastric epithelial cells and subsequently undergoes tyrosine phosphorylation. The phosphorylated CagA specifically binds SHP-2 phosphatase, activates the phosphatase activity, and thereby induces morphological transformation of cells. CagA proteins of most Western H. pylori isolates have a 34-amino acid sequence that variably repeats among different strains. Here, we show that the repeat sequence contains a tyrosine phosphorylation site. CagA proteins having more repeats were found to undergo greater tyrosine phosphorylation, to exhibit increased SHP-2 binding, and to induce greater morphological changes. In contrast, predominant CagA proteins specified by H. pylori strains isolated in East Asia, where gastric carcinoma is prevalent, had a distinct tyrosine phosphorylation sequence at the region corresponding to the repeat sequence of Western CagA. This East Asian-specific sequence conferred stronger SHP-2 binding and morphologically transforming activities to Western CagA. Finally, a critical amino acid residue that determines SHP-2 binding activity among different CagA proteins was identified. Our results indicate that the potential of individual CagA to perturb host-cell functions is determined by the degree of SHP-2 binding activity, which depends in turn on the number and sequences of tyrosine phosphorylation sites. The presence of distinctly structured CagA proteins in Western and East Asian H. pylori isolates may underlie the strikingly different incidences of gastric carcinoma in these two geographic areas.

Deregulation of SHP2 is associated with malignant diseases as well as developmental disorders. Although SHP2 is required for full activation of RAS signaling, other potential roles in cell physiology have not been elucidated. Here we show that SHP2 dephosphorylates parafibromin/Cdc73, a core component of the RNA polymerase II-associated factor (PAF) complex. Parafibromin is known to act as a tumor suppressor that inhibits cyclin D1 and c-myc by recruiting SUV39H1 histone methyltransferase. However, parafibromin can also act in the opposing direction by binding β-catenin, thereby activating pro-mitogenic/oncogenic Wnt signaling. We found that, upon tyrosine dephosphorylation by SHP2, parafibromin acquires the ability to stably bind β-catenin. The parafibromin/β-catenin interaction overrides parafibromin/SUV39H1-mediated transrepression and induces expression of Wnt target genes, including cyclin D1 and c-myc. Hence, SHP2 governs the opposing functions of parafibromin, deregulation of which may cause the development of tumors or developmental malformations.

Regulated activity of the retrograde molecular motor, cytoplasmic dynein, is crucial for multiple biological activities, and failure to regulate this activity can result in neuronal migration retardation or neuronal degeneration. The activity of dynein is controlled by the LIS1–Ndel1–Nde1 protein complex that participates in intracellular transport, mitosis, and neuronal migration. These biological processes are subject to tight multilevel modes of regulation. Palmitoylation is a reversible posttranslational lipid modification, which can dynamically regulate protein trafficking. We found that both Ndel1 and Nde1 undergo palmitoylation in vivo and in transfected cells by specific palmitoylation enzymes. Unpalmitoylated Ndel1 interacts better with dynein, whereas the interaction between Nde1 and cytoplasmic dynein is unaffected by palmitoylation. Furthermore, palmitoylated Ndel1 reduced cytoplasmic dynein activity as judged by Golgi distribution, VSVG and short microtubule trafficking, transport of endogenous Ndel1 and LIS1 from neurite tips to the cell body, retrograde trafficking of dynein puncta, and neuronal migration. Our findings indicate, to the best of our knowledge, for the first time that Ndel1 palmitoylation is a new mean for fine-tuning the activity of the retrograde motor cytoplasmic dynein.

Infection with cagA-positive Helicobacter pylori (H. pylori) is associated with atrophic gastritis, peptic ulcer, and gastric adenocarcinoma. The cagA gene product CagA is translocated from H. pylori into gastric epithelial cells and undergoes tyrosine phosphorylation by Src family kinases (SFKs). Tyrosine-phosphorylated CagA binds and activates SHP-2 phosphatase and the C-terminal Src kinase (Csk) while inducing an elongated cell shape termed the “hummingbird phenotype.” Here we show that CagA reduces the level of focal adhesion kinase (FAK) tyrosine phosphorylation in gastric epithelial cells. The decrease in phosphorylated FAK is due to SHP-2-mediated dephosphorylation of FAK at the activating phosphorylation sites, not due to Csk-dependent inhibition of SFKs, which phosphorylate FAK. Coexpression of constitutively active FAK with CagA inhibits induction of the hummingbird phenotype, whereas expression of dominant-negative FAK elicits an elongated cell shape characteristic of the hummingbird phenotype. These results indicate that inhibition of FAK by SHP-2 plays a crucial role in the morphogenetic activity of CagA. Impaired cell adhesion and increased motility by CagA may be involved in the development of gastric lesions associated with cagA-positive H. pylori infection.

Helicobacter pylori (H. pylori) is a causative agent of gastric diseases ranging from gastritis to cancer. The CagA protein is the product of the cagA gene carried among virulent H. pylori strains and is associated with severe disease outcomes, most notably gastric carcinoma. CagA is injected from the attached H. pylori into gastric epithelial cells and undergoes tyrosine phosphorylation. The phosphorylated CagA binds and activates SHP-2 phosphatase and thereby induces a growth factor-like morphological change termed the "hummingbird phenotype." In this work, we demonstrate that CagA is also capable of interacting with C-terminal Src kinase (Csk). As is the case with SHP-2, Csk selectively binds tyrosine-phosphorylated CagA via its SH2 domain. Upon complex formation, CagA stimulates Csk, which in turn inactivates the Src family of protein-tyrosine kinases. Because Src family kinases are responsible for CagA phosphorylation, an essential prerequisite of CagA.SHP-2 complex formation and subsequent induction of the hummingbird phenotype, our results indicate that CagA-Csk interaction down-regulates CagA.SHP-2 signaling by both competitively inhibiting CagA.SHP-2 complex formation and reducing levels of CagA phosphorylation. We further demonstrate that CagA.SHP-2 signaling eventually induces apoptosis in AGS cells. Our results thus indicate that CagA-Csk interaction prevents excess cell damage caused by deregulated activation of SHP-2. Attenuation of CagA activity by Csk may enable cagA-positive H. pylori to persistently infect the human stomach for decades while avoiding excess CagA toxicity to the host.

Helicobacter pylori CagA protein is associated with severe gastritis and gastric carcinoma. CagA is injected from the attached Helicobacter pylori into host cells and undergoes tyrosine phosphorylation. Wild-type but not phosphorylation-resistant CagA induced a growth factor–like response in gastric epithelial cells. Furthermore, CagA formed a physical complex with the SRC homology 2 domain (SH2)–containing tyrosine phosphatase SHP-2 in a phosphorylation-dependent manner and stimulated the phosphatase activity. Disruption of the CagA–SHP-2 complex abolished the CagA-dependent cellular response. Conversely, the CagA effect on cells was reproduced by constitutively active SHP-2. Thus, upon translocation, CagA perturbs cellular functions by deregulating SHP-2.

Palmitoylation is a key post-translational modification mediated by a family of DHHC-containing palmitoyl acyl-transferases (PATs). Unlike other lipid modifications, palmitoylation is reversible and thus often regulates dynamic protein interactions. We find that the mouse hair loss mutant, depilated, (dep) is due to a single amino acid deletion in the PAT, Zdhhc21, resulting in protein mislocalization and loss of palmitoylation activity. We examined expression of Zdhhc21 protein in skin and find it restricted to specific hair lineages. Loss of Zdhhc21 function results in delayed hair shaft differentiation, at the site of expression of the gene, but also leads to hyperplasia of the interfollicular epidermis (IFE) and sebaceous glands, distant from the expression site. The specific delay in follicle differentiation is associated with attenuated anagen propagation and is reflected by decreased levels of Lef1, nuclear β-catenin, and Foxn1 in hair shaft progenitors. In the thickened basal compartment of mutant IFE, phospho-ERK and cell proliferation are increased, suggesting increased signaling through EGFR or integrin-related receptors, with a parallel reduction in expression of the key differentiation factor Gata3. We show that the Src-family kinase, Fyn, involved in keratinocyte differentiation, is a direct palmitoylation target of Zdhhc21 and is mislocalized in mutant follicles. This study is the first to demonstrate a key role for palmitoylation in regulating developmental signals in mammalian tissue homeostasis., Author Summary During embryonic development, growth and patterning are regulated at many levels. Signals that mediate transcriptional activity, where and when genes are expressed, are a primary level of regulation. However, developmental signals can be further fine-tuned by modulating protein stability, localization, and activity via post-translational modifications. One such modification is the reversible addition of the fatty acid palmitate to proteins. This modification mediates dynamic trafficking of target proteins to specific subdomains of the cell. A large family of enzymes carries out this palmitoylation process, where each family member has specificity towards particular targets. However, the functional significance of palmitoylation during mammalian development is unclear. We present evidence of a critical role for palmitoylation during mouse development using a mutation of a specific palmitoylating enzyme, whose loss of function leads to hair loss and skin defects in depilated (dep) mice. Despite its restricted expression in hair follicles, loss of function of this enzyme results in developmental defects in nearby structures. We show that palmitoylation plays an important regulatory role in hair growth and epidermal homeostasis.