Activation of the lysosome-associated p61Hck isoform triggers the biogenesis of podosomes

Haematopoietic cell kinase (Hck) is a protein tyrosine kinase of the Src family specifically expressed in phagocytes as two isoforms, p59Hck and p61Hck, present at the plasma membrane and lysosomes, respectively. We report that ectopic expression of a constitutively active mutant of p61Hck (p61Hck(ca)) triggered the de novo formation of actin-rich rings at the ventral face of the cells that we characterized as bona fide podosome rosettes, structures involved in cell migration. Their formation required the adaptor domains and the kinase activity of p61Hck, the integrity of microfilament and microtubule networks and concerted action of Cdc42, Rac and Rho. Podosome rosette formation was either abolished when p61Hck(ca) was readdressed from lysosomes to the cytosol or triggered when p59Hck(ca) was relocalized to lysosomes. Lysosomal markers were present at podosome rosettes. By stimulating exocytosis of p61Hck(ca) lysosomes with a calcium ionophore, the formation of podosome rosettes was enhanced. Interestingly, we confirm that, in human macrophages, Hck and lysosomal markers were present at podosomes which were spatially reorganized as clusters, a foregoing step to form rosettes, upon expression of p61Hck(ca). We propose that lysosomes, under the control of p61Hck, are involved in the biogenesis of podosomes, a key phenomenon in the migration of phagocytes.     

Expressing murine p56Hck<Superscript>ca</Superscript> promotes HeLa cells’ motility and invasion via triggering redistribution of F-actin and microtubules

Hck is the unique example among the Src PTKs to be expressed as two isoforms, which are generated by alternative translation. The two isoforms differs from each other by a 21 N-terminal amino acids sequence which supports myristoylation. Though it has been shown that these different acylation states govern the different subcellular localization of the isoforms and each Hck isoform could play a specific role, little study focus on the function of p56Hck. To investigated the role of p56Hck isoform in cell migration, GFP targeted p56Hck plasmid and its constitutively active form were constructed and transiently transfected into HeLa cells, F-actin staining and Indirect immunofluorescence for microtubules were then performed. Phagokinetic track motility assay and In vitro invasion assays were also investigated after transiently transfection respectively. In this study, we found ectopically expressing a constitutively active form of 56Hck will lead to membrane protrusion and F-actin reorganization in HeLa cells. Both 56Hck and its constitutive active form will lead to redistribution of microtubules and enhancement of cell motility and cell invasion. Hck inhibitor PP2 supplementation eliminated cell motility and cell invasion of p56Hck while PP3, a negative control of PP2 didn’t eliminate cell motility and cell invasion of p56Hck. It is indicated that enhanced cell motility and cell invasion in p56Hck ectopically expressed HeLa cells are the results of reorganization of F-actin and microtubules.     

p59Hck isoform induces F-actin reorganization to form protrusions of the plasma membrane in a Cdc42- and Rac-dependent manner

Hck is a protein kinase of the Src family specifically expressed in phagocytes as two isoforms, p59Hck and p61Hck, localized at the plasma membrane and lysosomes, respectively. Their individual involvement in functions ascribed to Hck, phagocytosis, cell migration, and lysosome mobilization, is still unclarified. To investigate the specific role of p59Hck, a constitutively active variant in fusion with green fluorescent protein (p59Hck(ca)) was expressed in HeLa cells. p59Hck(ca) was found at focal adhesion sites and triggered reorganization of the actin cytoskeleton, leading to plasma membrane protrusions where it co-localized with F-actin. Similarly, microinjection of p59Hck(ca) cDNA in J774.A1 macrophages induced membrane protrusions. Whereas kinase activity and membrane association of p59Hck were dispensable for location at focal adhesions, p59Hck-induced membrane protrusions were dependent on kinase activity, plasma membrane association, and Src homology 2 but not Src homology 3 domain and were inhibited by dominant-negative forms of Cdc42 or Rac but not by blocking Rho activity. A dominant negative form of p59Hck inhibited the Cdc42- and Rac-dependent FcgammaRIIa-mediated phagocytosis. Expression of the Cdc42/Rac-interacting domain of p21-activated kinase in macrophages abolished the p59Hck(ca)-induced morphological changes. Therefore, p59Hck-triggered remodeling of the actin cytoskeleton depends upon the activity of Cdc42 and Rac to promote formation of membrane protrusions necessary for phagocytosis and cell migration.     

Hematopoietic cell kinase (Hck) isoforms and phagocyte duties – From signaling and actin reorganization to migration and phagocytosis

The activity of hematopoietic cell kinase (Hck), a member of the Src family kinases, is modulated by regulatory mechanisms leading to distinct protein conformations with gradual levels of activity. Hck is mostly expressed in phagocytes as two isoforms, p59Hck and p61Hck, which show distinct subcellular localizations and trigger distinct phenotypes when expressed ectopically in fibroblasts. Hck has been reported to be involved in phagocytosis, adhesion and migration, and to regulate formation of membrane protrusions, lysosome exocytosis, podosome formation, and actin polymerization. The present review focuses on the mechanisms regulating Hck activity as well as on the functions of Hck isoforms in phagocytes, and presents selected examples of Hck substrates and/or adaptors shown to interact with the kinase in myeloid cells. Deciphering Hck signaling pathways is a challenge to progress in the understanding of innate immune responses and pathologies involving phagocytes such as inflammatory diseases, leukemia, and human immunodeficiency virus-1 (HIV-1) infection.     

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.     

Hematopoietic cell kinase (Hck) isoforms and phagocyte duties - from signaling and actin reorganization to migration and phagocytosis

The activity of hematopoietic cell kinase (Hck), a member of the Src family kinases, is modulated by regulatory mechanisms leading to distinct protein conformations with gradual levels of activity. Hck is mostly expressed in phagocytes as two isoforms, p59Hck and p61Hck, which show distinct subcellular localizations and trigger distinct phenotypes when expressed ectopically in fibroblasts. Hck has been reported to be involved in phagocytosis, adhesion and migration, and to regulate formation of membrane protrusions, lysosome exocytosis, podosome formation, and actin polymerization. The present review focuses on the mechanisms regulating Hck activity as well as on the functions of Hck isoforms in phagocytes, and presents selected examples of Hck substrates and/or adaptors shown to interact with the kinase in myeloid cells. Deciphering Hck signaling pathways is a challenge to progress in the understanding of innate immune responses and pathologies involving phagocytes such as inflammatory diseases, leukemia, and human immunodeficiency virus-1 (HIV-1) infection.     

Activation of p61Hck triggers WASp- and Arp2/3-dependent actin-comet tail biogenesis and accelerates lysosomes

Secretory lysosomes exist in few cell types, but various mechanisms are involved to ensure their mobilization within the cytoplasm. In phagocytes, lysosome exocytosis is a regulated phenomenon at least in part under the control of the phagocyte-specific and lysosome-associated Src-kinase p61Hck (hematopoietic cell kinase). We show here that p61Hck activation triggered polymerization of actin at the membrane of lysosomes, which resulted in F-actin structures similar to comet tails observed on endocytic vesicles. We correlated this actin-comet biogenesis to a 35% acceleration of p61Hck-lysosomes in cells, which was dependent on actin polymerization and required an intact microtubular network. It was possible to initiate the formation of actin tails on p61Hck-positive lysosomes and on p61Hck-associated latex beads incubated in human phagocyte cytosolic extracts. The in vitro reconstitution on beads indicated that other lysosomal proteins were dispensable in this mechanism. The de novo actin polymerization process was functionally dependent on the kinase activity of Hck, WASp, the Arp2/3 complex, and Cdc42 but not Rac or Rho. Thus, we identified p61Hck as the first lysosomal protein able to recruit the molecular machinery responsible for actin tail formation. Altogether, our results suggest a new mechanism for lysosome motility involving p61Hck, actin-comet tail biogenesis, and the microtubule network.     

Palmitoylation of p59fyn is reversible and sufficient for plasma membrane association.

Members of the Src family of protein tyrosine kinases are localized to subspecialized regions of the plasma membrane. Herein we show that the N-terminal SH4 region of the Src family member p59fyn (Fyn) is both necessary and sufficient for targeting of Fyn and heterologous proteins to the plasma membrane and detergent-insoluble subdomains. Attachment of the first 16 amino acids of Fyn to a normally cytosolic protein, beta-galactosidase, resulted in distinct plasma membrane localization of the chimeric protein. Mutation of the palmitoylation site (cysteine-3) within Fyn16-beta-galactosidase or wild-type Fyn abrogated plasma membrane localization, resulting in redistribution of the mutant proteins into intracellular membranes. Substitution of the SH4 motif within Fyn with heterologous sequences from other palmitoylated proteins (G alpha o and GAP43) revealed that the presence of palmitate is sufficient to direct plasma membrane localization independent of surrounding amino acid sequences and myristate. Palmitoylated Fyn chimeras were also enriched in the Triton X-100-resistant matrix, whereas nonpalmitoylated forms of these proteins were detected in the detergent-soluble fraction. The palmitate moiety on Fyn exhibited a half-life of 1.5-2 h. In contrast, the half-life of the polypeptide backbone was 8 h, indicating that palmitoylation is a reversible modification. These studies establish that the palmitoylated SH4 sequence of Fyn can be used to specifically target proteins to the plasma membrane in a reversible manner.     

Human N-myristoyltransferases form stable complexes with lentiviral nef and other viral and cellular substrate proteins

Nef is a multifunctional virulence factor of primate lentiviruses that facilitates viral replication in the infected host. All known functions of Nef require that it be myristoylated at its N terminus. This reaction is catalyzed by N-myristoyltransferases (NMTs), which transfer myristate from myristoyl coenzyme A (myristoyl-CoA) to the N-terminal glycine of substrate proteins. Two NMT isoforms (NMT-1 and NMT-2) are expressed in mammalian cells. To provide a better mechanistic understanding of Nef function, we used biochemical and microsequencing techniques to isolate and identify Nef-associated proteins. Through these studies, NMT-1 was identified as an abundant Nef-associated protein. The Nef-NMT-1 complex is most likely a transient intermediate of the myristoylation reaction of Nef and is modulated by agents which affect the size of the myristoyl-CoA pool in the cell. We also examined two other proteins that bear an N-terminal myristoylation signal, human immunodeficiency virus type 1 Gag and Hck protein tyrosine kinase, and found that Gag bound preferentially the NMT-2 isoform, while Hck bound mostly to NMT-1. Recognition of different NMT isoforms by these viral and cellular substrate proteins suggests nonoverlapping roles for these enzymes in vivo and reveals a potential for the development of inhibitors that target the myristoylation of specific viral substrates more selectively.     

Subcellular localization and function of alternatively spliced Noxo1 isoforms

Nox organizer 1 (Noxo1), a p47(phox) homolog, is produced as four isoforms with unique N-terminal PX domains derived by alternative mRNA splicing. We compared the subcellular distribution of these isoforms or their isolated PX domains produced as GFP fusion proteins, as well as their ability to support Nox1 activity in several transfected models. Noxo1alpha, beta, gamma, and delta show different subcellular localization patterns, determined by their PX domains. In HEK293 cells, Noxo1beta exhibits prominent plasma membrane binding, Noxo1gamma shows plasma membrane and nuclear associations, and Noxo1alpha and delta localize primarily on intracellular vesicles or cytoplasmic aggregates, but not the plasma membrane. Nox1 activity correlates with Noxo1 plasma membrane binding in HEK293 cells, since Noxo1beta supports the highest activity and Noxo1gamma and Noxo1alpha support moderate or low activities, respectively. In COS-7 cells, where Noxo1alpha localizes on the plasma membrane, the activities supported by the three isoforms (alpha, beta, and gamma) do not differ significantly. The PX domains of beta and gamma bind the same phospholipids, including phosphatidic acid. These results indicate that the variant PX domains are unique determinants of Noxo1 localization and Nox1 function. Finally, the overexpressed Noxo1 isoforms do not affect p22(phox) localization, although Nox1 is needed to transport p22(phox) to the plasma membrane.     

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.     

The dually acylated NH2-terminal domain of gi1alpha is sufficient to target a green fluorescent protein reporter to caveolin-enriched plasma membrane domains. Palmitoylation of caveolin-1 is required for the recognition of dually acylated g-protein alpha subunits in vivo

Here we investigate the molecular mechanisms that govern the targeting of G-protein alpha subunits to the plasma membrane. For this purpose, we used Gi1alpha as a model dually acylated G-protein. We fused full-length Gi1alpha or its extreme NH2-terminal domain (residues 1-32 or 1-122) to green fluorescent protein (GFP) and analyzed the subcellular localization of these fusion proteins. We show that the first 32 amino acids of Gi1alpha are sufficient to target GFP to caveolin-enriched domains of the plasma membrane in vivo, as demonstrated by co-fractionation and co-immunoprecipitation with caveolin-1. Interestingly, when dual acylation of this 32-amino acid domain was blocked by specific point mutations (G2A or C3S), the resulting GFP fusion proteins were localized to the cytoplasm and excluded from caveolin-rich regions. The myristoylated but nonpalmitoylated (C3S) chimera only partially partitioned into caveolin-containing fractions. However, both nonacylated GFP fusions (G2A and C3S) no longer co-immunoprecipitated with caveolin-1. Taken together, these results indicate that lipid modification of the NH2-terminal of Gi1alpha is essential for targeting to its correct destination and interaction with caveolin-1. Also, a caveolin-1 mutant lacking all three palmitoylation sites (C133S, C143S, and C156S) was unable to co-immunoprecipitate these dually acylated GFP-G-protein fusions. Thus, dual acylation of the NH2-terminal domain of Gi1alpha and palmitoylation of caveolin-1 are both required to stabilize and perhaps regulate this reciprocal interaction at the plasma membrane in vivo. Our results provide the first demonstration of a functional role for caveolin-1 palmitoylation in its interaction with signaling molecules.     

N-terminal polybasic motifs are required for plasma membrane localization of Galpha(s) and Galpha(q)

Heterotrimeric G proteins typically localize at the cytoplasmic face of the plasma membrane where they interact with heptahelical receptors. For G protein alpha subunits, multiple membrane targeting signals, including myristoylation, palmitoylation, and interaction with betagamma subunits, facilitate membrane localization. Here we show that an additional membrane targeting signal, an N-terminal polybasic region, plays a key role in plasma membrane localization of non-myristoylated alpha subunits. Mutations of N-terminal basic residues in alpha(s) and alpha(q) caused defects in plasma membrane localization, as assessed through immunofluorescence microscopy and biochemical fractionations. In alpha(s), mutation of four basic residues to glutamine was sufficient to cause a defect, whereas in alpha(q) a defect in membrane localization was not observed unless nine basic residues were mutated to glutamine or if three basic residues were mutated to glutamic acid. betagamma co-expression only partially rescued the membrane localization defects; thus, the polybasic region is also important in the context of the heterotrimer. Introduction of a site for myristoylation into the polybasic mutants of alpha(s) and alpha(q) recovered strong plasma membrane localization, indicating that myristoylation and polybasic motifs may have complementary roles as membrane targeting signals. Loss of plasma membrane localization coincided with defects in palmitoylation. The polybasic mutants of alpha(s) and alpha(q) were still capable of assuming activated conformations and stimulating second messenger production, as demonstrated through GST-RGS4 interaction assays, cAMP assays, and inositol phosphate assays. Electrostatic interactions with membrane lipids have been found to be important in plasma membrane targeting of many proteins, and these results provide evidence that basic residues play a role in localization of G protein alpha subunits.     

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.     

Experimental testing of predicted myristoylation targets involved in asymmetric cell division and calcium-dependent signalling

Evolutionary conservation of N-terminal N-myristoylation within protein families indicates significant functional impact of this lipid posttranslational modification for function. In the MYRbase study (Maurer-Stroh et al. (2004) Genome Biology 5, R21), protein families with relevance to asymmetric cell division in animals and the group of plant calcium-dependent protein kinases (CPKs) have surfaced with many predicted myristoylated members. Here, we describe experimental in vitro verification of predicted myristoylation and explore its impact on subcellular localization for these targets in vivo. Our results confirm that, indeed, Numb isoform A, Neuralized isoforms C and D from Drosophila melanogaster and two Neuralized-like homologues from Mus musculus have the capability for N-terminal myristoylation in vitro and in vivo (in fly tissue and in mouse 3T3 cells respectively) whereas other isoforms such as Neuralized A and B have not. The latter two cases are an example of different potential of various isoforms for posttranslational modifications. Additionally, the Arabidopsis thaliana CDPKs CPK6, CPK9 and CPK13 are shown to be substrates for myristoylation in vitro, which also affects their subcellular localization (in Arabidopsis protoplasts and tobacco leaves). At the same time, CPK6 and CPK13 do not appear to be substrates of a NMT1-like enzyme; the reasons for differing substrate specificities of NMT homologues in plants are derived from the evolutionary divergence of their N-myristoyl transferase sequences. As a methodical advance, we describe a fast and very sensitive technique (compared to traditional autoradiography) for in vitro testing of myristoylation based on thin layer chromatography read-out of the incorporated radioactive myristoyl anchor with subsequent Western blotting detection for protein yield determination using the same membrane.     

Palmitoylation plays a role in targeting Vac8p to specific membrane subdomains

Vac8p is a multifunctional yeast protein involved in several distinct vacuolar events including vacuole inheritance, vacuole homotypic fusion, nucleus-vacuole junction formation and the cytoplasm to vacuole protein targeting pathway. Vac8p associates with the vacuole membrane via myristoylation and palmitoylation. Vac8p has three putative palmitoylation sites, at Cys 4, 5 and 7. Here, we show that each of these cysteines may serve as a palmitoylation site. Palmitoylation at Cys 7 alone provides partial function of Vac8p, whereas palmitoylation at either Cys 4 or Cys 5 alone is sufficient for Vac8p function. In the former mutant, there is a severe defect in the localization of Vac8p to the vacuole membrane, while in the latter mutants, there is a partial defect in the localization of Vac8p. In addition, our studies provide evidence that palmitoylation targets Vac8p to specific membrane subdomains.     

Sequence requirement for the nucleolar localization of human I-mfa domain-containing protein (HIC p40)

The human I-mfa domain-containing protein (HIC) mRNA produces two protein isoforms, HIC p32 and p40, synthesized from alternative translational initiations. p32 translation is initiated from a standard AUG codon and p40 is an N-terminal extension of p32 generated from an upstream GUG codon. The two isoforms show different subcellular localization: p32 is distributed throughout the cytoplasm whereas p40 can be found both in the cytoplasm and the nucleolus. To investigate the possibility that p40 contains a nucleolus targeting sequence in its N-terminal region, COS cells were transfected with an eukaryotic expression vector coding for green fluorescent protein (GFP) fused to the p40 N terminus. The localization of this fusion protein in the nucleolus indicated that the N-terminal amino acids of p40 probably contain a nucleolar localization signal (NoLS). To find the structural motifs required for nucleolar localization of p40, deletion mutants were expressed in COS cells as fusion polypeptides with GFP. We defined a domain of 19 amino acids near the N terminus that contains an arginine-rich subdomain that conforms to other known NoLS. To demonstrate that this sequence is an authentic NoLS, the sequence was fused to GFP. This fusion protein was observed to migrate into the nucleolus. Taken together, our studies demonstrate that p40 contains a NoLS.     

Vac8p, a Vacuolar Protein with Armadillo Repeats, Functions in both Vacuole Inheritance and Protein Targeting from the Cytoplasm to Vacuole

During each cell cycle, the yeast vacuole actively partitions between mother and daughter cells. This process requires actin, profilin, an unconventional myosin (Myo2p), and Vac8p. A mutant yeast strain, vac8, is defective in vacuole inheritance, specifically, in early vacuole migration. Vac8p is a 64-kD protein found on the vacuole membrane, a site consistent with its role in vacuole inheritance. Both myristoylation and palmitoylation are required for complete Vac8p localization. Interestingly, whereas myristoylation of Vac8p is not required for vacuole inheritance, palmitoylation is essential. Thus, palmitoylation appears to play a more direct role in vacuole inheritance. Most of the VAC8 sequence encodes 11 armadillo (Arm) repeats. Arm repeats are thought to mediate protein–protein interactions, and many Arm proteins have multiple functions. This is also true for Vac8p. In addition to its role in early vacuole inheritance, Vac8p is required to target aminopeptidase I from the cytoplasm to the vacuole. Mutant analysis demonstrates that Vac8p functions separately in these two processes. Vac8p cosediments with actin filaments. Vac8p is related to β-catenin and plakoglobin, which connect a specific region of the plasma membrane to the actin cytoskeleton. In analogy, Vac8p may link the vacuole to actin during vacuole partitioning.     

Multiple features of the p59fyn src homology 4 domain define a motif for immune-receptor tyrosine-based activation motif (ITAM) binding and for plasma membrane localization

The src family tyrosine kinase p59fyn binds to a signaling motif contained in subunits of the TCR known as the immune-receptor tyrosine- based activation motif (ITAM). This is a specific property of p59fyn because two related src family kinases, p60src and p56lck, do not bind to ITAMs. In this study, we identify the residues of p59fyn that are required for binding to ITAMs. We previously demonstrated that the first 10 residues of p59fyn direct its association with the ITAM. Because this region of src family kinases also directs their fatty acylation and membrane association (Resh, M.D. 1993, Biochim. Biophys. Acta 1155:307-322; Resh, M.D. 1994. Cell. 76:411-413), we determined whether fatty acylation and membrane association of p59fyn correlates with its ability to bind ITAMs. Four residues (Gly2, Cys3, Lys7, and Lys9) were required for efficient binding of p59fyn to the TCR. Interestingly, the same four residues are present in p56lyn, the other src family tyrosine kinase known to bind to the ITAM, suggesting that this set of residues constitutes an ITAM recognition motif. These residues were also required for efficient fatty acylation (myristoylation at Gly2 and palmitoylation at Cys3), and plasma membrane targeting of p59fyn. Thus, the signals that direct p59fyn fatty acylation and plasma membrane targeting also direct its specific ability to bind to TCR proteins.