The N-terminal SH4 region of the Src family kinase Fyn is modified by methylation and heterogeneous fatty acylation: role in membrane targeting, cell adhesion, and spreading

The N-terminal SH4 domain of Src family kinases is responsible for promoting membrane binding and plasma membrane targeting. Most Src family kinases contain an N-terminal Met-Gly-Cys consensus sequence that undergoes dual acylation with myristate and palmitate after removal of methionine. Previous studies of Src family kinase fatty acylation have relied on radiolabeling of cells with radioactive fatty acids. Although this method is useful for verifying that a given fatty acid is attached to a protein, it does not reveal whether other fatty acids or other modifying groups are attached to the protein. Here we use matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry to identify fatty acylated species of the Src family kinase Fyn. Our results reveal that Fyn is efficiently myristoylated and that some of the myristoylated proteins are also heterogeneously S-acylated with palmitate, palmitoleate, stearate, or oleate. Furthermore, we show for the first time that Fyn is trimethylated at lysine residues 7 and/or 9 within its N-terminal region. Both myristoylation and palmitoylation were required for methylation of Fyn. However, a general methylation inhibitor had no inhibitory effect on myristoylation and palmitoylation of Fyn, suggesting that methylation occurs after myristoylation and palmitoylation. Lysine mutants of Fyn that could not be methylated failed to promote cell adhesion and spreading, suggesting that methylation is important for Fyn function.     

A Calcium-Dependent Protein Kinase Is Systemically Induced upon Wounding in Tomato Plants

A full-length cDNA clone (LeCDPK1) from tomato (Lycopersicon esculentum) encoding a calcium-dependent protein kinase (CDPK) was isolated by screening a cDNA library from tomato cell cultures exposed to Cladosporium fulvum elicitor preparations. The predicted amino acid sequence of the cDNA reveals a high degree of similarity with other members of the CDPK family. LeCDPK1 has a putative N-terminal myristoylation sequence and presents a possible palmitoylation site. The in vitro translated protein conserves the biochemical properties of a member of the CDPK family. In addition, CDPK activity was detected in soluble and particulate extracts of tomato leaves. Basal levels of LeCDPK1 mRNA were detected by northern-blot analysis in roots, stems, leaves, and flowers of tomato plants. The expression of LeCDPK1 was rapidly and transiently enhanced in detached tomato leaves treated with pathogen elicitors and H2O2. Moreover, when tomato greenhouse plants were subjected to mechanical wounding, a transient increase of LeCDPK1 steady-state mRNA levels was detected locally at the site of the injury and systemically in distant non-wounded leaves. The increase observed in LeCDPK1 mRNA upon wounding correlates with an increase in the amount and in the activity of a soluble CDPK detected in extracts of tomato leaves, suggesting that this kinase is part of physiological plant defense mechanisms against biotic or abiotic attacks.     

N-terminally myristoylated Ras proteins require palmitoylation or a polybasic domain for plasma membrane localization.

Plasma membrane targeting of Ras requires CAAX motif modifications together with a second signal from an adjacent polybasic domain or nearby cysteine palmitoylation sites. N-terminal myristoylation is known to restore membrane binding to H-ras C186S (C-186 is changed to S), a mutant protein in which all CAAX processing is abolished. We show here that myristoylated H-ras C186S is a substrate for palmitoyltransferase, despite the absence of C-terminal farnesylation, and that palmitoylation is absolutely required for plasma membrane targeting of myristoylated H-ras. Similarly, the polybasic domain is required for specific plasma membrane targeting of myristoylated K-ras. In contrast, the combination of myristoylation plus farnesylation results in the mislocalization of Ras to numerous intracellular membranes. Ras that is only myristoylated does not bind with a high affinity to any membrane. The specific targeting of Ras to the plasma membrane is therefore critically dependent on signals that are contained in the hypervariable domain but can be supported by N-terminal myristoylation or C-terminal prenylation. Interestingly, oncogenic Ras G12V that is localized correctly to the plasma membrane leads to mitogen-activated protein kinase activation irrespective of the combination of targeting signals used for localization, whereas Ras G12V that is mislocalized to the cytosol or to other membranes activates mitogen-activated protein kinase only if the Ras protein is farnesylated.     

Cloning and biochemical properties of CDPK gene OsCDPK14 from rice

A rice CDPK gene, OsCDPK14 (AY144497), was cloned from developing caryopses of rice (Oryza sativa cv. Zhonghua 15). Its cDNA sequence (1922 bp) contains an ORF encoding a 514 amino acids protein (56.7kD, pl 5.18). OsCDPK14 shows the typical structural features of the CDPK family, including a conserved catalytic Ser/Thr kinase domain, an autoinhibitory domain and a CaM-like domain with four putative Ca2+-binding EF hands. Subcellular targeting indicated that OsCDPK14 was located in the cytoplasm, probably due to the absence of myristoylation and palmitoylation motifs. OsCDPK14 was expressed in Escherichia coli and purified from bacterial extracts. The recombinant protein was shown to be a functional protein kinase using Syntide-2, a synthetic peptide. Kinase activity was shown to be Ca2+-dependent, and this activation was strongly enhanced by Mn2+ and inhibited by W7 in vitro. These results provide significant insights into the regulation and biochemical properties of OsCDPK14, suggesting OsCDPK14 may be a signal factor of cytoplasm in rice plant.     

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.     

N-terminal motifs in some plant disease resistance proteins function in membrane attachment and contribute to disease resistance

To investigate the role of N-terminal domains of plant disease resistance proteins in membrane targeting, the N termini of a number of Arabidopsis and flax disease resistance proteins were fused to green fluorescent protein (GFP) and the fusion proteins localized in planta using confocal microscopy. The N termini of the Arabidopsis RPP1-WsB and RPS5 resistance proteins and the PBS1 protein, which is required for RPS5 resistance, targeted GFP to the plasma membrane, and mutation of predicted myristoylation and potential palmitoylation sites resulted in a shift to nucleocytosolic localization. The N-terminal domain of the membrane-attached Arabidopsis RPS2 resistance protein was targeted incompletely to the plasma membrane. In contrast, the N-terminal domains of the Arabidopsis RPP1-WsA and flax L6 and M resistance proteins, which carry predicted signal anchors, were targeted to the endomembrane system, RPP1-WsA to the endoplasmic reticulum and the Golgi apparatus, L6 to the Golgi apparatus, and M to the tonoplast. Full-length L6 was also targeted to the Golgi apparatus. Site-directed mutagenesis of six nonconserved amino acid residues in the signal anchor domains of L6 and M was used to change the localization of the L6 N-terminal fusion protein to that of M and vice versa, showing that these residues control the targeting specificity of the signal anchor. Replacement of the signal anchor domain of L6 by that of M did not affect L6 protein accumulation or resistance against flax rust expressing AvrL567 but removal of the signal anchor domain reduced L6 protein accumulation and L6 resistance, suggesting that membrane attachment is required to stabilize the L6 protein.     

Autophosphorylation and subcellular localization dynamics of a salt- and water deficit-induced calcium-dependent protein kinase from ice plant

A salinity and dehydration stress-responsive calcium-dependent protein kinase (CDPK) was isolated from the common ice plant (Mesembryanthemum crystallinum; McCPK1). McCPK1 undergoes myristoylation, but not palmitoylation in vitro. Removal of the N-terminal myristate acceptor site partially reduced McCPK1 plasma membrane (PM) localization as determined by transient expression of green fluorescent protein fusions in microprojectile-bombarded cells. Removal of the N-terminal domain (amino acids 1-70) completely abolished PM localization, suggesting that myristoylation and possibly the N-terminal domain contribute to membrane association of the kinase. The recombinant, Escherichia coli-expressed, full-length McCPK1 protein was catalytically active in a calcium-dependent manner (K0.5 = 0.15 microm). Autophosphorylation of recombinant McCPK1 was observed in vitro on at least two different Ser residues, with the location of two sites being mapped to Ser-62 and Ser-420. An Ala substitution at the Ser-62 or Ser-420 autophosphorylation site resulted in a slight increase in kinase activity relative to wild-type McCPK1 against a histone H1 substrate. In contrast, Ala substitutions at both sites resulted in a dramatic decrease in kinase activity relative to wild-type McCPK1 using histone H1 as substrate. McCPK1 undergoes a reversible change in subcellular localization from the PM to the nucleus, endoplasmic reticulum, and actin microfilaments of the cytoskeleton in response to reductions in humidity, as determined by transient expression of McCPK1-green fluorescent protein fusions in microprojectile-bombarded cells and confirmed by subcellular fractionation and western-blot analysis of 6x His-tagged McCPK1.     

A mouse brain cDNA encodes a novel protein with the protein kinase C phosphorylation site domain common to MARCKS

We have isolated a mouse brain cDNA clone encoding a protein of 200 amino acids (Mr 20,165) with partial homology with MARCKS (myristoylated alanine-rich C-kinase substrate). Two regions show similarity with MARCKS, one is the kinase C phosphorylation site domain which is supposed to bind calmodulin, and the other is the region near to the N-terminus, including the consensus sequence of myristoylation. It has a similar amino acid composition to MARCKS, but the content of alanine is not as high. It is distributed throughout the mouse brain, but the pattern is not identical with that of MARCKS. Both proteins may be members of a new protein family involved in coupling the protein kinase C and calmodulin signal transduction systems.     

CHRK1, a chitinase-related receptor-like kinase in tobacco

A cDNA encoding a chitinase-related receptor-like kinase, designated CHRK1, was isolated from tobacco (Nicotiana tabacum). The C-terminal kinase domain (KD) of CHRK1 contained all of the conserved amino acids of serine/threonine protein kinases. The putative extracellular domain was closely related to the class V chitinase of tobacco and to microbial chitinases. CHRK1 mRNA accumulation was strongly stimulated by infection with fungal pathogen and tobacco mosaic virus. Amino acid-sequence analysis revealed that the chitinase-like domain of CHRK1 lacked the essential glutamic acid residue required for chitinase activity. The recombinant chitinase-like domain did not show any catalytic activity for either oligomeric or polymeric chitin substrates. The recombinant KD of CHRK1 exhibited autophosphorylation, but the mutant KD with a mutation in the essential ATP-binding site did not, suggesting that CHRK1 encoded a functional kinase. CHRK1 was detected in membrane fractions of tobacco BY2 cells. Furthermore, CHRK1-GFP fusion protein was localized in plasma membranes when it was expressed in animal cells. This is the first report of a new type of receptor-like kinase containing a chitinase-like sequence in the putative extracellular domain.     

Protein N-acylation overrides differing targeting signals

In a bioinformatics based screen for chloroplast-localized protein kinases we noticed that available protein targeting predictors falsely predicted chloroplast localization. This seems to be due to interference with N-terminal protein acylation, which is of particular importance for protein kinases. Their N-myristoylation was found to be highly overrepresented in the proteome, whereas myristoylation motifs are almost absent in known chloroplast proteins. However, only abolishing their myristoylation was not sufficient to target those kinases to chloroplasts and resulted in nuclear accumulation instead. In contrast, inhibition of N-myristoylation of a calcium-dependent protein kinase was sufficient to alter its localization from the plasma membrane to chloroplasts and chloroplast localization of ferredoxin-NADP+ reductase and Rubisco activase could be efficiently suppressed by artificial introduction of myristoylation and palmitoylation sites.     

Functional roles for fatty acylated amino-terminal domains in subcellular localization

Several membrane-associating signals, including covalently linked fatty acids, are found in various combinations at the N termini of signaling proteins. The function of these combinations was investigated by appending fatty acylated N-terminal sequences to green fluorescent protein (GFP). Myristoylated plus mono/dipalmitoylated GFP chimeras and a GFP chimera containing a myristoylated plus a polybasic domain were localized similarly to the plasma membrane and endosomal vesicles, but not to the nucleus. Myristoylated, nonpalmitoylated mutant chimeric GFPs were localized to intracellular membranes, including endosomes and the endoplasmic reticulum, and were absent from the plasma membrane, the Golgi, and the nucleus. Dually palmitoylated GFP was localized to the plasma membrane and the Golgi region, but it was not detected in endosomes. Nonacylated GFP chimeras, as well as GFP, showed cytosolic and nuclear distribution. Our results demonstrate that myristoylation is sufficient to exclude GFP from the nucleus and associate with intracellular membranes, but plasma membrane localization requires a second signal, namely palmitoylation or a polybasic domain. The similarity in localization conferred by the various myristoylated and palmitoylated/polybasic sequences suggests that biophysical properties of acylated sequences and biological membranes are key determinants in proper membrane selection. However, dual palmitoylation in the absence of myristoylation conferred significant differences in localization, suggesting that multiple palmitoylation sites and/or enzymes may exist.     

Mutants of the protein serine kinase PSKH1 disassemble the Golgi apparatus

We have dissected the molecular determinants involved in targeting the protein serine kinase PSKH1 to the endoplasmic reticulum (ER), the Golgi apparatus, and the plasma membrane (PM). Given this intracellular localization pattern, a potential role of PSKH1 in the secretory pathway was explored. The amino-terminal of PSKH1 revealed a striking similarity to the often acylated Src homology domain 4 (SH4)-harboring nonreceptor tyrosine kinases. Biochemical studies demonstrated that PSKH1 is myristoylated on glycine 2 and palmitoylated on cysteine 3. Dual amino-terminal acylation targets PSKH1 to Golgi as shown by colocalization with beta-COP and GM130, while nonpalmitoylated (myristoylated only) PSKH1 targets intracellular membranes colocalizing with protein disulphide isomerase (PDI, a marker for ER). Immunoelectron microscopy revealed that the dually acylated amino-terminal domain (in fusion with EGFP) was targeted to Golgi membranes as well as to the plasma membrane (PM), suggesting that the amino-terminal domain provides PSKH1 with membrane specificity dependent on its fatty acylation status. Subcellular fractionation by sucrose gradient analysis confirmed the impact of dual fatty acylation on endomembrane targeting, while cytosol and membrane fractioning revealed that myristoylation but not palmitoylation was required for general membrane association. A minimal region required for proper Golgi targeting of PSKH1 was identified within the first 29 amino acids. Expression of a PSKH1 mutant where the COOH-terminal kinase domain was swapped with green fluorescent protein and cysteine 3 was exchanged with serine resulted in disassembly of the Golgi apparatus as visualized by redistribution of beta-COP and GM130 to a diffuse cytoplasmic pattern, while leaving the tubulin skeleton intact. Our results suggest a structural and regulatory role of PSKH1 in maintenance of the Golgi apparatus, a key organelle within the secretory pathway.     

Interaction with Gbetagamma is required for membrane targeting and palmitoylation of Galpha(s) and Galpha(q)

Peripheral membrane proteins utilize a variety of mechanisms to attach tightly, and often reversibly, to cellular membranes. The covalent lipid modifications, myristoylation and palmitoylation, are critical for plasma membrane localization of heterotrimeric G protein alpha subunits. For alpha(s) and alpha(q), two subunits that are palmitoylated but not myristoylated, we examined the importance of interacting with the G protein betagamma dimer for their proper plasma membrane localization and palmitoylation. Conserved alpha subunit N-terminal amino acids predicted to mediate binding to betagamma were mutated to create a series of betagamma binding region mutants expressed in HEK293 cells. These alpha(s) and alpha(q) mutants were found in soluble rather than particulate fractions, and they no longer localized to plasma membranes as demonstrated by immunofluorescence microscopy. The mutations also inhibited incorporation of radiolabeled palmitate into the proteins and abrogated their signaling ability. Additional alpha(q) mutants, which contain these mutations but are modified by both myristate and palmitate, retained their localization to plasma membranes and ability to undergo palmitoylation. These findings identify binding to betagamma as a critical membrane attachment signal for alpha(s) and alpha(q) and as a prerequisite for their palmitoylation, while myristoylation can restore membrane localization and palmitoylation of betagamma binding-deficient alpha(q) subunits.