Palmitoylation of stathmin family proteins domain A controls Golgi versus mitochondrial subcellular targeting

Background information. Precise localization of proteins to specialized subcellular domains is fundamental for proper neuronal development and function. The neural microtubule‐regulatory phosphoproteins of the stathmin family are such proteins whose specific functions are controlled by subcellular localization. Whereas stathmin is cytosolic, SCG10, SCLIP and RB3/RB3′/RB3″ are localized to the Golgi and vesicle‐like structures along neurites and at growth cones. We examined the molecular determinants involved in the regulation of this specific subcellular localization in hippocampal neurons in culture. Results. We show that their conserved N‐terminal domain A carrying two palmitoylation sites is dominant over the others for Golgi and vesicle‐like localization. Using palmitoylation‐deficient GFP (green fluorescent protein) fusion mutants, we demonstrate that domains A of stathmin proteins have the particular ability to control protein targeting to either Golgi or mitochondria, depending on their palmitoylation. This regulation involves the co‐operation of two subdomains within domain A, and seems also to be under the control of its SLD (stathmin‐like domain) extension. Conclusions. Our results unravel that, in specific biological conditions, palmitoylation of stathmin proteins might be able to control their targeting to express their functional activities at appropriate subcellular sites. They, more generally, open new perspectives regarding the role of palmitoylation as a signalling mechanism orienting proteins to their functional subcellular compartments.     

Specific serine-proline phosphorylation and glycogen synthase kinase 3β-directed subcellular targeting of stathmin 3/Sclip in neurons

During nervous system development, neuronal growth, migration, and functional morphogenesis rely on the appropriate control of the subcellular cytoskeleton including microtubule dynamics. Stathmin family proteins play major roles during the various stages of neuronal differentiation, including axonal growth and branching, or dendritic development. We have shown previously that stathmins 2 (SCG10) and 3 (SCLIP) fulfill distinct, independent and complementary regulatory roles in axonal morphogenesis. Although the two proteins have been proposed to display the four conserved phosphorylation sites originally identified in stathmin 1, we show here that they possess distinct phosphorylation sites within their specific proline-rich domains (PRDs) that are differentially regulated by phosphorylation by proline-directed kinases involved in the control of neuronal differentiation. ERK2 or CDK5 phosphorylate the two proteins but with different site specificities. We also show for the first time that, unlike stathmin 2, stathmin 3 is a substrate for glycogen synthase kinase (GSK) 3β both in vitro and in vivo. Interestingly, stathmin 3 phosphorylated at its GSK-3β target site displays a specific subcellular localization at neuritic tips and within the actin-rich peripheral zone of the growth cone of differentiating hippocampal neurons in culture. Finally, pharmacological inhibition of GSK-3β induces a redistribution of stathmin 3, but not stathmin 2, from the periphery toward the Golgi region of neurons. Stathmin proteins can thus be either regulated locally or locally targeted by specific phosphorylation, each phosphoprotein of the stathmin family fulfilling distinct and specific roles in the control of neuronal differentiation.     

Stathmin family proteins display specific molecular and tubulin binding properties

Stathmin family phosphoproteins (stathmin, SCG10, SCLIP, and RB3/RB3'/RB3") are involved in signal transduction and regulation of microtubule dynamics. With the exception of stathmin, they are expressed exclusively in the nervous system, where they display different spatio-temporal and functional regulations and hence play at least partially distinct and possibly complementary roles in relation to the control of development, plasticity, and neuronal activities. At the molecular level, each possesses a specific "stathmin-like domain" and, with the exception of stathmin, various combinations of N-terminal extensions involved in their association with intracellular membrane compartments. We show here that each stathmin-like domain also displays specific biochemical and tubulin interaction properties. They are all able to sequester two alpha/beta tubulin heterodimers as revealed by their inhibitory action on tubulin polymerization and by gel filtration. However, they differ in the stabilities of the complexes formed as well as in their interaction kinetics with tubulin followed by surface plasmon resonance as follows: strong stability and slow kinetics for RB3; medium for SCG10, SCLIP, and stathmin; and weak stability and rapid kinetics for RB3'. These results suggest that the fine-tuning of their stathmin-like domains contributes to the specific functional roles of stathmin family proteins in the regulation of microtubule dynamics within the various cell types and subcellular compartments of the developing or mature nervous system.     

Fluorescence-based methods to image palmitoylated proteins

A well known function of palmitoylation is to promote protein binding to cell membranes. Until recently, it was unclear what additional roles, if any, palmitoylation has in controlling protein localization in cells. Recent studies of palmitoylated forms of the small GTPase Ras have now revealed that palmitoylation plays multiple roles in the regulation of protein trafficking, including targeting proteins into the secretory pathway and recycling proteins between the plasma membrane and Golgi complex. We here describe how quantitative fluorescence microscopy and photobleaching approaches can be used to study the intracellular targeting and trafficking of GFP-tagged palmitoylated proteins in living cells. We discuss (1) general considerations for fluorescence recovery after photobleaching (FRAP) measurements of GFP-tagged proteins; (2) FRAP-based assays to test the strength of binding of palmitoylated proteins to cell membranes; (3) methods to establish the kinetics and mechanisms of recycling of palmitoylated proteins between the Golgi complex and the plasma membrane; (4) the use of the palmitoylation inhibitor 2-bromo-palmitate as a tool to study the dynamic regulation of protein targeting and trafficking by palmitate turnover.     

Intrinsic Signals in the Unique Domain Target p56lckto the Plasma Membrane Independently of CD4

In T lymphocytes, the Src-family protein tyrosine kinase p56^lck (Lck) is mostly associated with the cytoplasmic face of the plasma membrane. To determine how this distribution is achieved, we analyzed the location of Lck in lymphoid and in transfected nonlymphoid cells by immunofluorescence. We found that in T cells Lck was targeted correctly, independently of the cell surface proteins CD4 and CD8 with which it interacts. Similarly, in transfected NIH-3T3 fibroblasts, Lck was localized at the plasma membrane, indicating that T cell–specific proteins are not required for targeting. Some variation in subcellular distribution was observed when Lck was expressed in HeLa and MDCK cells. In these cells, Lck associated with both the plasma membrane and the Golgi apparatus, while subsequent expression of CD4 resulted in the loss of Golgi-associated staining. Together, these data indicate that Lck contains intrinsic signals for targeting to the plasma membrane. Furthermore, delivery to this site may be achieved via association with exocytic transport vesicles.

A mutant Lck molecule in which the palmitoylation site at cysteine 5 was changed to lysine (LC2) localized to the plasma membrane and the Golgi region in NIH3T3 cells. However, the localization of a mutant in which the palmitoylation site at cysteine 3 was changed to serine (LC1) was indistinguishable from wild-type Lck. Chimeras composed of only the unique domain of Lck linked to either c-Src or the green fluorescent protein similarly localized to the plasma membrane of NIH-3T3 cells. Thus, the targeting of Lck appears to be determined primarily by its unique domain and may be influenced by the use of different palmitoylation sites.

     

Palmitoylation of GAP-43 by the ER-Golgi intermediate compartment and Golgi apparatus.

Palmitoylation of the neuronal plasticity protein GAP-43 has previously been shown to occur at the plasma membrane, but the site of initial palmitoylation has not been identified. To identify this organelle we have incubated GAP-43 with various subcellular fractions and have analyzed palmitoylation by the Triton X-114 partitioning method. In vitro-translated [(35)S]methionine-labeled GAP-43 was incubated with plasma membrane, nuclei, mitochondria, Golgi apparatus and a rough microsome preparation that contained the ER-Golgi intermediate compartment (ERGIC), but not plasma membrane or Golgi apparatus. GAP-43 partitioned into Triton X-114 in the presence of plasma membrane, Golgi, and ERGIC membranes, but not nuclei or mitochondria. Partitioning caused by the ERGIC was blocked by pretreatment of the membranes with the palmitoylation inhibitors dithiothreitol, tunicamycin, and low temperature, and by treatment of GAP-43 with iodoacetamide. The time course of partitioning agreed closely with the time course of incorporation of radioactive palmitate into proteins as reported previously. Because the ERGIC has a broad distribution in the cell, our results provide evidence that the ERGIC is the initial site of GAP-43 palmitoylation.     

A proline-rich region and nearby cysteine residues target XLalphas to the Golgi complex region

XLalphas is a splice variant of the heterotrimeric G protein, Galpha(s), found on Golgi membranes in cells with regulated and constitutive secretion. We examined the role of the alternatively spliced amino terminus of XLalphas for Golgi targeting with the use of subcellular fractionation and fluorescence microscopy. XLalphas incorporated [(3)H]palmitate, and mutation of cysteines in a cysteine-rich region inhibited this incorporation and lessened membrane attachment. Deletion of a proline-rich region abolished Golgi localization of XLalphas without changing its membrane attachment. The proline-rich and cysteine-rich regions together were sufficient to target the green fluorescent protein, a cytosolic protein, to Golgi membranes. The membrane attachment and Golgi targeting of the fusion protein required the putative palmitoylation sites, the cysteine residues in the cysteine-rich region. Several peripheral membrane proteins found at the Golgi have proline-rich regions, including a Galpha(i2) splice variant, dynamin II, betaIII spectrin, comitin, and a Golgi SNARE, GS32. Our results suggest that proline-rich regions can be a Golgi-targeting signal for G protein alpha subunits and possibly for other peripheral membrane proteins as well.     

Non-Microtubular Localizations of Microtubule-Associated Protein 6 (MAP6)

MAP6 proteins (MAP6s), which include MAP6-N (also called Stable Tubule Only Polypeptide, or STOP) and MAP6d1 (MAP6 domain-containing protein 1, also called STOP-Like protein 21 kD, or SL21), bind to and stabilize microtubules. MAP6 deletion in mice severely alters integrated brain functions and is associated with synaptic defects, suggesting that MAP6s may also have alternative cellular roles. MAP6s reportedly associate with the Golgi apparatus through palmitoylation of their N-terminal domain, and specific isoforms have been shown to bind actin. Here, we use heterologous systems to investigate several biochemical properties of MAP6 proteins. We demonstrate that the three N-terminal cysteines of MAP6d1 are palmitoylated by a subset of DHHC-type palmitoylating enzymes. Analysis of the subcellular localization of palmitoylated MAP6d1, including electron microscopic analysis, reveals possible localization to the Golgi and the plasma membrane but no association with the endoplasmic reticulum. Moreover, we observed localization of MAP6d1 to mitochondria, which requires the N-terminus of the protein but does not require palmitoylation. We show that endogenous MAP6d1 localized at mitochondria in mature mice neurons as well as at the outer membrane and in the intermembrane space of purified mouse mitochondria. Last, we found that MAP6d1 can multimerize via a microtubule-binding module. Interestingly, most of these properties of MAP6d1 are shared by MAP6-N. Together, these results describe several properties of MAP6 proteins, including their intercellular localization and multimerization activity, which may be relevant to neuronal differentiation and synaptic functions.     

Proteomic profiling of S-acylated macrophage proteins identifies a role for palmitoylation in mitochondrial targeting of phospholipid scramblase 3

S-Palmitoylation, the reversible post-translational acylation of specific cysteine residues with the fatty acid palmitate, promotes the membrane tethering and subcellular localization of proteins in several biological pathways. Although inhibiting palmitoylation holds promise as a means for manipulating protein targeting, advances in the field have been hampered by limited understanding of palmitoylation enzymology and consensus motifs. In order to define the complement of S-acylated proteins in the macrophage, we treated RAW 264.7 macrophage membranes with hydroxylamine to cleave acyl thioesters, followed by biotinylation of newly exposed sulfhydryls and streptavidin-agarose affinity chromatography. Among proteins identified by LC-MS/MS, S-acylation status was established by spectral counting to assess enrichment under hydroxylamine versus mock treatment conditions. Of 1183 proteins identified in four independent experiments, 80 proteins were significant for S-acylation at false discovery rate = 0.05, and 101 significant at false discovery rate = 0.10. Candidate S-acylproteins were identified from several functional categories, including membrane trafficking, signaling, transporters, and receptors. Among these were 29 proteins previously biochemically confirmed as palmitoylated, 45 previously reported as putative S-acylproteins in proteomic screens, 24 not previously associated with palmitoylation, and three presumed false-positives. Nearly half of the candidates were previously identified by us in macrophage detergent-resistant membranes, suggesting that palmitoylation promotes lipid raft-localization of proteins in the macrophage. Among the candidate novel S-acylproteins was phospholipid scramblase 3 (Plscr3), a protein that regulates apoptosis through remodeling the mitochondrial membrane. Palmitoylation of Plscr3 was confirmed through (3)H-palmitate labeling. Moreover, site-directed mutagenesis of a cluster of five cysteines (Cys159-161-163-164-166) abolished palmitoylation, caused Plscr3 mislocalization from mitochondrion to nucleus, and reduced macrophage apoptosis in response to etoposide, together suggesting a role for palmitoylation at this site for mitochondrial targeting and pro-apoptotic function of Plscr3. Taken together, we propose that manipulation of protein palmitoylation carries great potential for intervention in macrophage biology via reprogramming of protein localization.     

Akr1p-dependent palmitoylation of Yck2p yeast casein kinase 1 is necessary and sufficient for plasma membrane targeting

The Yck2 protein is a plasma membrane-associated casein kinase 1 isoform that attaches to membranes via palmitoylation of its C terminus. We have demonstrated that Yck2p traffics to the plasma membrane on secretory vesicles. Because Akr1p, the palmitoyl transferase for Yck2p, is located on Golgi membranes, it is likely that Yck2p first associates with Golgi membranes, and then is somehow recruited to budding plasma membrane-destined vesicles. We show here that residues 499-546 are sufficient for minimal Yck2p palmitoylation and plasma membrane localization. We previously described normal plasma membrane targeting of a Yck2p construct with the final five amino acids of Ras2p substituting for the final two Cys residues of Yck2p. This Yck2p variant no longer requires Akr1p for membrane association, but targets normally. We have generated the C-terminal deletions previously shown to affect Yck2p membrane association in this variant to determine which residues are important for targeting and/or modification. We find that all of the sequences previously identified as important for plasma membrane association are required only for Akr1p-dependent modification. Furthermore, palmitoylation is sufficient for specific association of Yck2p with secretory vesicles destined for the plasma membrane. Finally, both C-terminal Cys residues are palmitoylated, and dual acylation is required for efficient membrane association.     

The palmitoyl transferase DHHC2 targets a dynamic membrane cycling pathway: regulation by a C-terminal domain

Intracellular palmitoylation dynamics are regulated by a large family of DHHC (Asp-His-His-Cys) palmitoyl transferases. The majority of DHHC proteins associate with endoplasmic reticulum (ER) or Golgi membranes, but an interesting exception is DHHC2, which localizes to dendritic vesicles of unknown origin in neurons, where it regulates dynamic palmitoylation of PSD95. Dendritic targeting of newly synthesized PSD95 is likely preceded by palmitoylation on Golgi membranes by DHHC3 and/or DHHC15. The precise intracellular distribution of DHHC2 is presently unclear, and there is very little known in general about how DHHC proteins achieve their respective localizations. In this study, membrane targeting of DHHC2 in live and fixed neuroendocrine cells was investigated and mutational analysis employed to define regions of DHHC2 that regulate targeting. We report that DHHC2 associates with the plasma membrane, Rab11-positive recycling endosomes, and vesicular structures. Plasma membrane integration of DHHC2 was confirmed by labeling of an extrafacial HA epitope in nonpermeabilized cells. Antibody-uptake experiments suggested that DHHC2 traffics between the plasma membrane and intracellular membranes. This dynamic localization was confirmed using fluorescence recovery after photo-bleaching analysis, which revealed constitutive refilling of the recycling endosome (RE) pool of DHHC2. The cytoplasmic C-terminus of DHHC2 regulates membrane targeting and a mutant lacking this domain was associated with the ER. Although DHHC2 is closely related to DHHC15, these proteins populate distinct membrane compartments. Construction of chimeric DHHC2/DHHC15 proteins revealed that this difference in localization is a consequence of divergent sequences within their C-terminal tails. This study is the first to highlight dynamic cycling of a mammalian DHHC protein between clearly defined membrane compartments, and to identify domains that specify membrane targeting of this protein family.     

SNAP-25 Palmitoylation and Plasma Membrane Targeting Require a Functional Secretory Pathway

Synaptosomal-associated protein of 25 kDa (SNAP-25) is a palmitoylated membrane protein essential for neurotransmitter release from synaptic terminals. We used neuronal cell lines to study the biosynthesis and posttranslational processing of SNAP-25 to investigate how palmitoylation contributes to the subcellular localization of the protein. SNAP-25 was synthesized as a soluble protein that underwent palmitoylation approximately 20 min after synthesis. Palmitoylation of the protein coincided with its stable membrane association. Treatment of cells with brefeldin A or other disrupters of transport inhibited palmitoylation of newly synthesized SNAP-25 and abolished membrane association. These results demonstrate that the processing of SNAP-25 and its targeting to the plasma membrane depend on an intact transport mechanism along the exocytic pathway. The kinetics of SNAP-25 palmitoylation and membrane association and the sensitivity of these parameters to brefeldin A suggest a novel trafficking pathway for targeting proteins to the plasma membrane. In vitro, SNAP-25 stably associated with membranes was not released from the membrane after chemical deacylation. We propose that palmitoylation of SNAP-25 is required for initial membrane targeting of the protein but that other interactions can maintain membrane association in the absence of fatty acylation.     

Lack of palmitoylation redirects p59Hck from the plasma membrane to p61Hck-positive lysosomes

Hck, a protein-tyrosine kinase of phagocytes, is the unique member of the Src family expressed under two alternatively translated isoforms differing in their N-terminal site of acylation: p61(Hck) has an additional 21-amino acid sequence comprising a single myristoylation motif, whereas p59(Hck) N terminus has myristoylation and palmitoylation sites. To identify the molecular determinants involved in the targeting of each isoform, they were fused to GFP and expressed in HeLa and CHO cells. p61(Hck) was associated with lysosomal vesicles, whereas p59(Hck) was found at the plasma membrane and to a low extent associated with lysosomes. Their unique N-terminal domains were sufficient to target GFP to the corresponding intracellular compartments. Mutation of the palmitoylation site of p59(Hck) redirected this isoform to lysosomes, indicating that the palmitoylation state governs the association of p59(Hck) with the plasma membrane or with lysosomes. In addition, both isoforms and the nonpalmitoylated p59(Hck) mutant were found on the Golgi apparatus, suggesting a role of this organelle in the subcellular sorting of Hck isoforms. Regarding their subcellular localizations, we propose that bi-acylated p59(Hck) might transduce plasma membrane receptor signals, whereas p61(Hck) and the nonpalmitoylated p59(Hck) might control the biogenesis of phagolysosomes, two functions yet proposed for Hck in phagocytes.     

Palmitoylated Ras proteins traffic through recycling endosomes to the plasma membrane during exocytosis

Ras proteins regulate cell growth, death, and differentiation, and it is well established that this functional versatility is accomplished through their different subcellular localizations. Palmitoylated H- and N-Ras are believed to localize at the perinuclear Golgi and plasma membrane (PM). Notably, however, recycling endosomes (REs) also localize to a perinuclear region, which is often indistinguishable from the Golgi. In this study, we show that active palmitoylated Ras proteins mainly localize intracellularly at REs and that REs act as a way station along the post-Golgi exocytic pathway to the PM. H-Ras requires two palmitoyl groups for RE targeting. The lack of either or both palmitoyl groups leads to the mislocalization of the mutant proteins to the endoplasmic reticulum, Golgi apparatus, or the PM. Therefore, we demonstrate that palmitoylation directs Ras proteins to the correct intracellular organelles for trafficking and activity.     

Ndel1 palmitoylation: a new mean to regulate cytoplasmic dynein activity

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.     

The First 35 Amino Acids and Fatty Acylation Sites Determine the Molecular Targeting of Endothelial Nitric Oxide Synthase into the Golgi Region of Cells: A Green Fluorescent Protein Study

Catalytically active endothelial nitric oxide synthase (eNOS) is located on the Golgi complex and in the caveolae of endothelial cells (EC). Mislocalization of eNOS caused by mutation of the N-myristoylation or cysteine palmitoylation sites impairs production of stimulated nitric oxide (NO), suggesting that intracellular targeting is critical for optimal NO production. To investigate the molecular determinants of eNOS targeting in EC, we constructed eNOS–green fluorescent protein (GFP) chimeras to study its localization in living and fixed cells. The full-length eNOS–GFP fusion colocalized with a Golgi marker, mannosidase II, and retained catalytic activity compared to wild-type (WT) eNOS, suggesting that the GFP tag does not interfere with eNOS localization or function. Experiments with different size amino-terminal fusion partners coupled to GFP demonstrated that the first 35 amino acids of eNOS are sufficient to target GFP into the Golgi region of NIH 3T3 cells. Additionally, the unique (Gly-Leu)₅ repeat located between the palmitoylation sites (Cys-15 and -26) of eNOS is necessary for its palmitoylation and thus localization, but not for N-myristoylation, membrane association, and NOS activity. The palmitoylation-deficient mutants displayed a more diffuse fluorescence pattern than did WT eNOS–GFP, but still were associated with intracellular membranes. Biochemical studies also showed that the palmitoylation-deficient mutants are associated with membranes as tightly as WT eNOS. Mutation of the N-myristoylation site Gly-2 (abolishing both N-myristoylation and palmitoylation) caused the GFP fusion protein to distribute throughout the cell as GFP alone, consistent with its primarily cytosolic nature in biochemical studies. Therefore, eNOS targets into the Golgi region of NIH 3T3 cells via the first 35 amino acids, including N-myristoylation and palmitoylation sites, and its overall membrane association requires N-myristoylation but not cysteine palmitoylation. These results suggest a novel role for fatty acylation in the specific compartmentalization of eNOS and most likely, for other dually acylated proteins, to the Golgi complex.     

Modulation of neuronal protein trafficking and function by palmitoylation

Modification of proteins with the lipid palmitate regulates targeting to specific vesicular compartments and synaptic membranes. Mounting evidence indicates that this lipid modification modulates diverse aspects of neuronal development and synaptic transmission. In particular, palmitoylation regulates the function of proteins that control neuronal differentiation, axonal pathfinding and filopodia formation. In addition, trafficking of numerous proteins associated with synaptic vesicle release machinery requires protein palmitoylation. Remarkably, reversible palmitoylation of specific scaffolding proteins and signaling molecules dynamically regulates ion channel clustering and synaptic strength. The recent discovery of enzymes that palmitoylate specific subsets of synaptic proteins suggests that this process is tightly controlled in neurons.     

WAVE2 targeting to phosphatidylinositol 3,4,5-triphosphate mediated by insulin receptor substrate p53 through a complex with WAVE2

Membrane targeting of WAVE2 along microtubules to phosphatidylinositol 3,4,5-triphosphate (PIP₃) in response to an extracellular stimulus requires Rac1, Pak1, stathmin, and EB1. However, whether WAVE2 interacts directly with PIP₃ or not remains unclear. We demonstrate that insulin-like growth factor I (IGF-I) induces WAVE2 membrane targeting, accompanied by phosphorylation of Pak1 at serine 199/204 (Ser199/204) and stathmin at Ser38 in the inner cytoplasmic region. This is spatially independent of the membrane region where the IGF-I receptor (IGF-IR) is locally activated. WAVE2, phosphorylated Pak1, and phosphorylated stathmin located at the microtubule ends began to accumulate at the leading edge of cells in close proximity to PIP₃ that was produced in a phosphatidylinositol 3-kinase (PI 3-kinase)-dependent manner. The PIP₃-beads binding assay revealed that insulin receptor substrate p53 (IRSp53) and actin rather than WAVE2 bound to PIP₃. IRSp53 constitutively associated with WAVE2 and these two proteins colocalized with PIP₃ at the leading edge after IGF-I stimulation. Suppression of IRSp53 expression by two independent small interfering RNAs (siRNAs) completely inhibited IGF-I-induced membrane targeting and local accumulation of WAVE2 at the leading edge of cells. We propose that IRSp53 constitutively forms a complex with WAVE2 and is crucial for membrane targeting followed by local accumulation of WAVE2 at the leading edge of cells through linking WAVE2 to PIP₃ that is produced near locally activated IGF-IR in response to IGF-I.     

H-ras but not K-ras traffics to the plasma membrane through the exocytic pathway

Ras proteins must be localized to the inner surface of the plasma membrane to be biologically active. The motifs that effect Ras plasma membrane targeting consist of a C-terminal CAAX motif plus a second signal comprising palmitoylation of adjacent cysteine residues or the presence of a polybasic domain. In this study, we examined how Ras proteins access the cell surface after processing of the CAAX motif is completed in the endoplasmic reticulum (ER). We show that palmitoylated CAAX proteins, in addition to being localized at the plasma membrane, are found throughout the exocytic pathway and accumulate in the Golgi region when cells are incubated at 15 degrees C. In contrast, polybasic CAAX proteins are found only at the cell surface and not in the exocytic pathway. CAAX proteins which lack a second signal for plasma membrane targeting accumulate in the ER and Golgi. Brefeldin A (BFA) significantly inhibits the plasma membrane accumulation of newly synthesized, palmitoylated CAAX proteins without inhibiting their palmitoylation. BFA has no effect on the trafficking of polybasic CAAX proteins. We conclude that H-ras and K-ras traffic to the cell surface through different routes and that the polybasic domain is a sorting signal diverting K-Ras out of the classical exocytic pathway proximal to the Golgi. Farnesylated Ras proteins that lack a polybasic domain reach the Golgi but require palmitoylation in order to traffic further to the cell surface. These data also indicate that a Ras palmitoyltransferase is present in an early compartment of the exocytic pathway.     

Palmitoylation by DHHC5/8 targets GRIP1 to dendritic endosomes to regulate AMPA-R trafficking

Palmitoylation, a key regulatory mechanism controlling protein targeting, is catalyzed by DHHC-family palmitoyl acyltransferases (PATs). Impaired PAT activity is linked to neurodevelopmental and neuropsychiatric disorders, suggesting critical roles for palmitoylation in neuronal function. However, few substrates for specific PATs are known, and functional consequences of palmitoylation events are frequently uncharacterized. Here, we identify the closely related PATs DHHC5 and DHHC8 as specific regulators of the PDZ domain protein GRIP1b. Binding, palmitoylation, and dendritic targeting of GRIP1b require a PDZ ligand unique to DHHC5/8. Palmitoylated GRIP1b is targeted to trafficking endosomes and may link endosomes to kinesin motors. Consistent with this trafficking role, GRIP1b's palmitoylation turnover rate approaches the highest of all reported proteins, and palmitoylation increases GRIP1b's ability to accelerate AMPA-R recycling. To our knowledge, these findings identify the first neuronal DHHC5/8 substrate, define novel mechanisms controlling palmitoylation specificity, and suggest further links between dysregulated palmitoylation and neuropathological conditions.