Signaling through Src-family kinases (SFKs) in hematopoietic cells

SFKs play initiating and critical roles in signaling through surface receptors on hematopoietic cells. Five SFKs primarily are expressed in leukocytes: Blk, Fgr, Hck, Lyn and Lck (Bolen and Brugge, 1997). The most well-studied immunoreceptors utilizing SFKs are the antigen receptors on B- and T lymphocytes (BCRs and TCRs), the receptor for IgE (FcRI) on the surface of mast cells, basophils and eosinophils and the receptors for IgG (FcRI, FcRIIA, FcRIII) on the surface of macrophages, monocytes, myeloid, NK, and mast cells (Takai, 2002; Veillette et al., 2002). Engagement of antigen receptors results in the immediate activation of the SFKs Lyn/Fyn/Blk (BCR) and Lck/Fyn (TCR). Myeloid cells on the other hand primarily express Hck, Fgr and Lyn and these SFKs respond not only to FcR clustering (Takai, 2002) but also to integrin engagement (Lowell et al., 1996). In cases involving antigen receptors and FcRs, activated SFKs phosphorylate tyrosine residues present in immune receptor tyrosine-based activation motifs (ITAMs) in the signaling subunits of the receptor (Cambier, 1995). As described elsewhere in this issue, signals emanating from ITAMs generate phosphorylation-based signaling cascades that ultimately result in the activation of transcription factors necessary for the proliferation and/or differentiation of immune cells.

Strategies for modulating SFKs

Like many signaling molecules, SFKs are composed of a series of modular domains. Individual domains regulate cellular localization (SH4), interaction with binding partners (SH2 and SH3) and enzymatic activity (SH1). All SFKs are N-terminally myristoylated and seven out of nine family members are also N-terminally palmitoylated (Wolven et al., 1997). Each of these domains provides a potential point of interdiction as interference with any domain significantly compromises the ability of SFKs to mediate signaling cascades. The critical role that SFKs play in hematopoietic signaling dictates that these tyrosine kinases be tightly regulated. Thus, immune cells have evolved various strategies to control SFK function. The enzymatic activities of SFKs are regulated by tyrosine phosphorylation, with the phosphorylation of a conserved tyrosine in the activation loop generating an active form of the enzyme, and phosphorylation of a conserved tyrosine in the C-terminal tail resulting in an intramolecular binding event causing inactivation (Sicheri et al., 1997; Xu et al., 1997; Harrison, 2003). Activation loop phosphorylation is mediated by the SFKs themselves, while the C-terminal Src kinase (Csk) and family member Chk are responsible for the inhibitory phosphorylation (Veillette et al., 2002). Since the kinases are regulated by tyrosine phosphorylation, tyrosine phosphatases also are major contributors to the regulation of kinase activity. Several excellent reviews on the role of tyrosine phosphatases in immune signaling have been published recently (Veillette et al., 2002; Hermiston et al., 2003; Mustelin et al., 2003) and discussion here will be limited to a few specific examples. Recently, a new mode of influencing SFK function has emerged that is focused on submembrane location and is regulated by both lipid modifications and interactions with localized binding partners. Thus, the relocalization of the SFKs themselves, as well as the kinases and phosphatases responsible for regulating their catalytic activity is turning out to be a major mechanism of controlling SFK function. For the experimentalist, pharmacological agents targeted to important SFK functional domains or covalent modifications can be used to artificially alter the activity or localization of SFKs as a means of studying their function in immune cells.

Inhibitors of catalytic activity

By far the most popular approach to the chemical interdiction of SFKs is the use of small molecule inhibitors targeted to the catalytic domain. To be useful, inhibitors must be cell permeable, exhibit specificity or selectivity for the target of interest and be sufficiently potent to compete for the target in the presence of an excess of the endogenous ligand, which, for kinase inhibitors, is typically ATP. For research purposes, the most commonly used inhibitors of SFKs are the pyrazolopyrimidines, PP1 (1) and PP2 (2) (Hanke et al., 1996) (Figure 1). These were developed originally as probes for the study of T-cell signaling as a means of differentiating between processes dependent on Lck or Fyn from those dependent on cytoplasmic tyrosine kinases of other families. This utility is based on their considerable selectivity for SFKs. For example, PP1 and PP2 inhibit Lck and Fyn in vitro with IC₅₀ values in the range of 4–6 nM as compared to IC₅₀ values of greater than 50 M for Zap-70 and Jak2. In intact cells, PP1 and PP2 exhibit half-maximal inhibition of signaling from the T-cell receptor for antigen (TCR) at a concentration of 500 nM.

Figure 1.

Chemical structures of SFK inhibitors

Full figure and legend (31K)

X-ray crystallographic studies indicate that PP1 and PP2 bind to SFKs in a cleft between the N- and C-terminal lobes that is normally occupied by ATP (Schindler et al., 1999; Zhu et al., 1999). The pyrazolopyrimidine ring makes two of the same hydrogen bonding contacts, one to the backbone carbonyl of Glu-317 and one to the backbone amide of Met-310, which are made by the adenine ring of the nonhydrolyzable analog of ATP, AMPPNP (Schindler et al., 1999; Zhu et al., 1999). The selectivity of PP1 and PP2 for SFKs, however, is a property of the methylphenyl or chlorophenyl side chains, which access a hydrophobic binding pocket adjacent to the ATP-binding site (Liu et al., 1999; Schindler et al., 1999; Zhu et al., 1999). This is a site that is not occupied by the nucleotide. While the presence of this hydrophobic pocket is not unique to SFKs, the topology of the pocket varies among different kinase families and access to it is restricted by amino-acid side chains of varying sizes. In all SFKs, a Thr residue (Thr-338 of Hck or Thr-316 of Lck) allows access to the pocket. For most other kinases, larger residues at this position, such as the Met found in Zap-70, restrict access rendering them insensitive to inhibition by PP1 or PP2. Interestingly, a point mutation that resulted in the replacement of Thr-338 by Ile has rendered v-Src less sensitive than c-Src to inhibition by PP1 (Liu et al., 1999).

The presence of a Thr at the position analogous to Src amino acid 338 is not restricted entirely to SFKs. For example, the platelet-derived growth factor receptor (PDGFR) and c-Abl both have Thr at this position and, as a consequence, exhibit a significant sensitivity to PP1 and PP2 (Blake et al., 1999; Liu et al., 1999; Waltenberger et al., 1999). Efforts to identify an inhibitor selective for SFKs without crossreactivity to the PDGFR led to the development of the indolinone, SU6656 (6) (Blake et al., 2000). SU6656 is essentially inactive as an inhibitor of PDGFR, but is a potent inhibitor of c-Src, Fyn, Yes and Lyn, but not of Lck (although another study analysing the selectivity of protein kinase inhibitors indicated good potency for SU6656 against Lck (Bain et al., 2003). SU6656, although highly selective for SFKs, does exhibit some crossreactivity with certain protein serine/threonine kinases such as RSK, AMPK and phosphorylase kinase (Bain et al., 2003). However, this pattern of crossreactivity is distinct from that of PP1 and PP2. Thus, a process or signaling event that is inhibited potently by either PP1 or PP2 and also by SU6656 can reasonably be assumed to be dependent on the activity of an SFK.

The presence of the hydrophobic binding pocket revealed by analysis of the structures of Hck and Lck complexed with PP1 or PP2 has been exploited by Shokat and co-workers (Bishop et al., 2000) in a clever strategy for the creation of mutant kinases engineered to be selective targets for analogs of PP1 and PP2 modified with side chains too bulky to be accommodated within the binding pockets of wild-type kinases. If the methylphenyl side chain of PP1 is replaced with a larger naphthyl or naphthylmethyl group (4,5), the resulting compounds are poor inhibitors of wild-type protein kinases of any class including v-Src and Fyn. However, if the residue restricting access to the hydrophobic binding pocket is changed to a smaller residue, such as Gly or Ala, the resulting mutant kinase becomes exquisitely sensitive to the C3-1'-naphthylmethyl or C3-1'-naphthyl PP1 analogs. For example, v-Src(I338G) and c-Fyn(T339G) are inhibited by the modified PP1 analogs with IC₅₀ values in the range of 1–6 nM, while the wild-type enzymes are inhibited by concentrations three orders of magnitude higher. This approach is generally applicable to protein kinases of diverse families. In systems that can be manipulated genetically to replace the endogenous kinase with an analog-sensitive mutant, these inhibitors can be used as 'chemical switches' to block selectively the activity of the mutant kinase so that its function can be evaluated unambiguously. Jurkat T cells deficient in Lck (JCaM1) (Straus and Weiss, 1992) or DT40 B cells deficient in Lyn (Takata et al., 1994) would be examples of cellular model systems in which this technology could easily be exploited.

An examination of the structures of PP1 bound to Hck in its inactive conformation and PP2 bound to Lck in its active conformation revealed that the size of the hydrophobic binding pocket varied depending on the activation state of the kinase (Schindler et al., 1999). PP1 leaves approximately 100 Å³ of the pocket unfilled in the inactive Hck kinase. However, the pocket is collapsed in the active Lck structure due to phosphorylation of the activation loop. This observation raised the possibility that PP1 could be redesigned to take advantage of this enlarged cavity to obtain inhibitors that selectively stabilize SFKs in their inactive conformations. Consistent with this hypothesis, compounds related to PP1 and PP2, but containing a side chain extended by addition of a 5-(4-phenoxyphenyl) substituent (3) are potent inhibitors of Lck that preferentially target the inactive, unphosphorylated kinase (Arnold et al., 2000). The resulting molecules are 100-fold more potent than PP1 at inhibiting TCR-stimulated IL-2 synthesis in Jurkat T cells. Interestingly, these compounds have been further elaborated to generate orally active agents capable of inhibiting IL-2 production in vivo (Burchat et al., 2002).

The next step will be the development of inhibitors selective for individual members of the Src-family. This will be a much more difficult task due to the high degree of sequence identity shared by family members. Thus, pharmacological approaches for distinguishing which of the several Src-family members is responsible for regulating a particular physiological response are not yet available, although progress is being made toward this goal. For example, BMS-243117 (7), which was identified using library screening and medicinal chemistry approaches (Das et al., 2003), is eightfold more potent as an inhibitor of Lck than Fyn, the next most sensitive Src-family member, and more than 200-fold more potent than Hck. Molecular modeling studies suggest that differences in amino-acid residues lining the ATP-binding pocket might underlie the selectivity. More exquisite selectivity is likely to require molecules that can access multiple binding sites. The recent identification of a selective c-Src inhibitor is a prime example of this type of approach (Maly et al., 2000). In this strategy, small molecule inhibitors of relatively low potency were selected from combinatorial libraries and then linked together to form additional libraries of coupled ligands composed of two distinct binding elements. Screening against c-Src yielded an inhibitor (8) that was inactive against Lck, and exhibited an 80-fold preference for c-Src as compared to Fyn, and a 200-fold preference as compared to Lyn. An extension of this technique to other SFK members might reasonably be expected to yield a series of reagents useful for studying the function of specific kinases in immune cells.

Inhibitors directed against the SH2 domain

In addition to the catalytic domain, the SH2 domain has received considerable attention as a target for small molecules that could interdict the function of the kinase in immune cells. This is a reasonable target since inactivation of the Lck SH2 domain abrogates TCR-dependent signaling in Jurkat T cells (Straus et al., 1996). The primary structural determinants that define the specificity of Src-family SH2 domain-phosphoprotein interactions lie within the three amino acids just distal to the phosphotyrosine residue. Phosphopeptides as short as four amino acids retain the ability to form tight and specific interactions (Gilmer et al., 1994). The screening of random phosphotyrosine-containing peptide libraries indicated that the sequence pYEEI is bound preferentially by Src family SH2 domains (Songyang et al., 1993). The three-dimensional structures of liganded Src family SH2 domains reveal two major binding pockets, one highly hydrophilic pocket occupied by the phosphotyrosine and a second hydrophobic pocket occupied by the pY+3 Ile residue (Eck et al., 1993; Waksman et al., 1993). The intervening glutamate residues lie on the surface and do not form extensive interactions with the SH2 domain. The binding interaction has been described as a 'two-pronged plug engaging a two-holed socket' (Kuriyan and Cowburn, 1997).

Investigations into the design of synthetic ligands specifically for the SH2 domain of Lck have shown that modifications to the pYEEI structure can be made that significantly enhance the affinity of the ligand for the domain. For example, addition of a coumarin moiety to the N-terminus of the peptide enhances binding by two orders of magnitude (Lee and Lawrence, 1999). Lawrence and co-workers have further used a parallel synthetic approach whereby each glutamyl residue was individually replaced with 2,3-diaminopropanoic acid to which any one of 1000 different carboxylic acids was attached (Yeh et al., 2001; Lawrence, 2003). By combining active leads from the two combinatorial libraries, a di-substituted phosphopeptide (9) was generated that was bound by the Lck SH2 domain with a dissociation constant of 0.2 nM, more than four orders of magnitude tighter than pYEEI.

The use of phosphopeptides as ligands for probing biological systems, however, faces numerous challenges, not the least of which are delivery across biological membranes and stability to intracellular phosphatases and proteases. However, the attractiveness of SH2 domains as targets has spurred considerable activity into the development of design strategies to circumvent these problems. The peptide backbone can be eliminated through the construction of small molecule, peptidomimetic and nonpeptide inhibitors that have been rationally designed to contain pharmacophores separated by an appropriate spacer to allow occupancy of both the phosphotyrosine and hydrophobic binding pockets (Sawyer et al., 2002). The phosphotyrosine residue itself can be replaced by mimics containing phosphonates, phosphinates or one or more carboxylates that are insensitive to phosphotyrosine phosphatase activity (Burke and Lee, 2003; Sundaramoorthi et al., 2003). Furthermore, the anionic nature of the phosphate or phosphonate can be masked through the formation of esters that allow penetration of the compound through biological membranes (Stankovic et al., 1997; Rickles et al., 1998). These advances should allow, in the near future, ready access to stable inhibitors designed to penetrate cells and target the SH2 domains of hematopoietic cell kinases that will be as useful to investigators as inhibitors targeted to the catalytic domains. In fact, by coupling small molecule inhibitors together by the appropriate linker, it may even be possible to target both the domains simultaneously (Profit et al., 2001).

Interdiction by sequestration

Lipid rafts

Aside from direct inhibition of enzymatic activity by cellular kinases and phosphatases (e.g. phosphorylation by Csk/Chk, dephosphorylation by PEP, SHP-1, possibly CD45), SFK function is regulated by subcellular localization dictated both by localized binding partners and lipid-directed microenvironments. During the last 5 years, the literature has exploded with information regarding the role of 'lipid rafts' in immunoreceptor signaling and several excellent reviews focused on microdomains and immune signaling have been published (Ilangumaran et al., 2000; Janes et al., 2000; Langlet et al., 2000; Bi and Altman, 2001; Germain, 2001; Holowka and Baird, 2001; Leitenberg et al., 2001; Miceli et al., 2001; Dykstra et al., 2003; Hoøejí, 2003; Pizzo and Viola, 2003; Harder and Engelhardt, 2004). The majority of these reviews summarize the case for SFK-dependent immune receptor signaling either initiating or being sustained (or both) in defined plasma membrane microdomains, although cautionary thoughts were voiced early on (Germain, 2001). Lipid rafts represent specialized, liquid-ordered patches of the lipid bilayer maintained by thermodynamic-directed packing of glycospingolipids, glycerophospholipids with saturated acyl chains and GPI-anchored proteins (GPI-AP) between planar molecules of cholesterol. These ordered patches are in contrast to the rest of the bilayer, which is envisioned as relatively disordered. SFKs along with other signaling molecules are thought to either constitutively reside in lipid rafts targeted there by their myristoyl and palmitoyl groups or relocalize to lipid rafts following immunoreceptor engagement (Filipp et al., 2003). Extensive studies support this organized and localization-directed view of signaling from surface receptors on T and B cells and mast cells. The model is an exciting and satisfying view of SFK-initiated signaling as it provides for the spatial as well as temporal sequestration of SFKs either away from or concentrated in close proximity to regulating kinases and phosphatases as well downstream targets.

A major problem with the lipid raft model is that lipid rafts cannot be 'seen' by standard optical microscopy leading to the current thinking that rafts are quite small, within the 5–70 nM range (Anderson and Jacobson, 2002; Sharma et al., 2004). Moreover, the biochemical detergent-based methods used to isolate lipid rafts were reported to induce their formation (Heerklotz, 2002). It is now abundantly clear that the distinction between raft-resident and nonresident signaling molecules is dependent on the detergent conditions used (Schuck et al., 2003). These issues have raised serious questions regarding the lipid raft-based model of SFK-mediated signaling (Germain, 2001; Munro, 2003). Attempts to circumvent technical limitations resulted in the generation of novel approaches including FRET analysis (Varma and Mayor, 1998; Kenworthy et al., 2000; Zacharias et al., 2002; Sharma et al., 2004) and single particle/molecule tracking (Pralle et al., 2000; Schultz et al., 2000; Dietrich et al., 2002; Vrljic et al., 2002). Reflective of the results obtained with these more sophisticated microscopy techniques, two very recent studies employing FRET analysis of GPI-AP proteins in live cells arrive at divergent, although not totally incompatible views. Glebov and Nichols (2004) find no evidence for the clustering (i.e. colocalization) of putative raft-resident proteins in the membranes of either resting or activated T cells, seriously contesting the view that lipid rafts are large, stable structures harboring multiple T-cell signaling molecules (Pierce, 2004). A somewhat different conclusion was reached by Sharma et al. (2004) who found evidence for organized nanoscale GPI-AP-containing, cholesterol-sensitive clusters. In addition, Wilson et al. (2000) find evidence for small clusters of FcRI and Lyn on the cytoplasmic face of membrane sheets derived from unstimulated RBL-2H3 cells. It would appear that if lipid rafts do exist in immune cells, they are likely to be small entities that can accommodate at most a few proteins in the resting state. Based on the fact that unlike many raft-resident proteins, SFKs appear in lipid rafts independent of the detergent used for their isolation, these kinases may be represented in these small raft entities.

Whether one is a raft believer or a raft agnostic, it seems likely that the lipid modifications (palmitic and myristic acid) on SFKs play a critical role in their correct membrane localization relative to their regulators and substrates and that lipid-driven clustering in membrane microdomains in immune cells occurs at some level. One of the puzzling features of SFKs is their relatively equal distribution between detergent insoluble fractions (the classic definition of lipid rafts) and the rest of the membrane (Hawash et al., 2002a, 2002b). This may be important, as evidence exists that the two pools of SFKs are differentially regulated. The SFK Lck, for example, is activated outside of lipid rafts in T cells, but rapidly translocates into rafts where it is necessary for the activation of Fyn (Filipp et al., 2003). Raft-associated Lyn on the other hand appears to be constitutively more active than the subset of Lyn found within the nonraft region of membrane in RBL-2H3 basophilic leukemia cells (Young et al., 2003). It has been proposed that crosslinking of the Fc receptor in this system promotes receptor phosphorylation by localizing crosslinked FcRI with active Lyn in lipid rafts (Young et al., 2003).

Several mechanisms can be envisioned to explain the differential distribution of SFKs between lipid rafts and nonraft regions of the membrane. As palmitoylation is a reversible event (Paige et al., 1993) and necessary for the localization of SFKs to lipid rafts (Shenoy-Scaria et al., 1994; Kabouridis et al., 1997; Wolven et al., 1997), raft-excluded SFKs may be depalmitoylated. Consistent with this idea, palmitoyltransferase activity has been reported to be enriched in raft microdomains (Dunphy et al., 2001). Therefore palmitoylation/depalmitoylation events may play important roles in the interdiction of SFKs by sequestration. Following receptor-ligation, activated SFKs are targeted for degradation by their interaction with the ubiquitin E3 ligase, Cbl (Panchamoorthy et al., 1996; Tezuka et al., 1996; Andoniou et al., 2000; Rao et al., 2002b). In resting cells, Cbl is found exclusively in the cytosol and nonraft regions of the membrane (Lafont and Simons, 2001; Hawash et al., 2002a, 2002b). Following activation of RBL-2H3 cells, Cbl was reported to translocate to lipid rafts where it colocalized with Lyn (Lafont and Simons, 2001). The interaction of SFKs with Cbl is complex involving SFK SH3 and SH2 domains, as well as the activating phosphotyrosine residue in the activation loop (Rao et al., 2002a). In resting hematopoietic cells, Cbl constitutively associates with the SH3 domains of SFKs (e.g. Lck, Fyn, Lyn) and for Lck, the association takes place outside of lipid rafts (Hawash et al., 2002a, 2002b). Moreover, overexpression of Cbl in resting Jurkat T cells results in a translocation of Lck from lipid rafts to the nonraft regions of the membrane (Hawash et al., 2002a, 2002b). Thus, by binding to SFKs, Cbl may interdict their function through: (1) physically sequestering them away from lipid rafts in resting cells and (2) mediating their degradation in activated cells. It also has been suggested that Lck is maintained outside lipid rafts through its association with the T-cell coreceptor CD4 (Filipp et al., 2003).

Inhibitors of protein acylation

The importance of the proper addressing of SFKs to their appropriate intracellular locations suggests that chemical interventions can be designed to interdict SFK function in immune cells. Most SFKs are both myristoylated and palmitoylated on amino-acid residues in the SH4 domain. N-myristoylation occurs during protein synthesis and is catalysed by myristoyl-CoA : protein N-myristoyltransferase (NMT), which recognizes as substrates myristoyl-CoA and a nascent polypeptide chain bearing a free N-terminal glycine followed by a restricted set of possible NMT recognition sequences (Towler et al., 1987; Giang and Cravatt, 1998; Farazi et al., 2001; Maurer-Stroh et al., 2002). S-Palmitoylation, in contrast, is a reversible, post-translational modification most likely catalysed by one or more palmitoyltransferases, examples of which have only recently been described (Linder and Deschenes, 2003). In this reaction, palmitate is transferred from palmitoyl-CoA to one or more cysteines located just distal to the N-terminal glycine (Shenoy-Scaria et al., 1993; Alland et al., 1994). Site-directed mutants lacking the acceptor glycine or cysteine residues are defective in both membrane localization and function (Resh, 1994). Thus, inhibitors or alternate substrates for protein acyltransferases can be used to modulate the properties of SFKs in hematopoietic cells.

The most commonly employed inhibitor of protein N-myristoylation is 2-hydroxymyristic acid (HMA) (10). HMA itself is a poor inhibitor of NMT, but is taken up by cells and converted by cellular acyl-CoA synthetases to 2-hydroxymyristoyl CoA, a potent (K_i=45 nM), dead-end inhibitor of NMT (Paige et al., 1990). Treatment of T cells with HMA leads to the synthesis of nonmyristoylated Lck, which is cytosolic and relatively unstable such that prolonged treatment can deplete the kinase from the cell (Nadler et al., 1993). Similarly, Fyn is relocalized to the soluble fraction in HMA-treated cells and is no longer able to associate with the TCR chain (van't Hof and Resh, 1999), and Lyn is displaced from lipid rafts, inhibiting FcRII phosphorylation and clustering in monocytic cells (Kwiatkowska et al., 2003). HMA is a selective inhibitor of protein myristoylation and does not block palmitoylation, allowing an independent analysis of the two processes (Paige et al., 1990).

A related approach can be used to block protein S-palmitoylation. Treatment of cells with 2-bromopalmitate inhibits protein acylation in general, but has a greater impact on palmitoylation than myristoylation (Webb et al., 2000). Under treatment conditions where the palmitoylation of Fyn was reduced by 90%, myristoylation was reduced by 70%. The mechanism of action of 2-bromopalmitate is not known. However, it is known that 2-bromomyristate does not block protein myristoylation (Paige et al., 1990) and 2-hydroxypalmitate does not block protein palmitoylation (Webb et al., 2000), suggesting fundamental differences in the mechanisms of how HMA and 2-bromopalmitate inhibit the two different acylation processes. The treatment of T cells with 2-bromopalmitate inhibits the localization of both Fyn and Lck to lipid rafts and, consequently, inhibits TCR-mediated signaling (Webb et al., 2000). Likewise, Lyn is displaced from lipid rafts in monocytic cells upon treatment with 2-bromopalmitate, inhibiting FcRII-mediated actin rearrangements (Kwiatkowska et al., 2003). Cerulenin, a general inhibitor of fatty acid synthase (Omura, 1976), additionally inhibits protein palmitoylation (Schlessinger and Malfer, 1982; Lawrence et al., 1999). Analogs of cerulenin have been prepared that preferentially block protein palmitoylation as compared to fatty acid synthesis (Lawrence et al., 1999; De Vos et al., 2001), but, to our knowledge, the effects of these on SFKs have not been examined.

An alternative approach to modulating the activity of SFKs is to use an alternate substrate for the acyltransferase rather than an inhibitor. The incorporation of a fatty acyl chain with modified properties, such as reduced hydrophobicity, can alter the properties of the acylated protein. Mass spectrometric analyses indicate that the SFK, Fyn, is modified on the N-terminal glycine almost exclusively by attachment of myristate (Liang et al., 2004). However, NMT will accept a fairly wide variety of fatty acid analogs if they are similar in overall length to myristate (Kishore et al., 1991; Devadas et al., 1992; Liang et al., 2001). Since myristate levels in cells are relatively low, supplying cells with an external source of an alternate substrate can lead to its incorporation into the protein in place of the naturally occurring fatty acid. For example, addition of the unsaturated fatty acids, 5-cis-tetradecenoic acid (14 : 1) and 5-cis,8-cis-tetradecadienoic acid (14 : 2), to cultured cells leads to the heterogeneous acylation of Fyn (Liang et al., 2001). Similarly, addition to cells of heteroatom-substituted myristate analogs with reduced hydrophobicities leads to the selective incorporation of compounds into subsets of acylated proteins, some of which redistribute from the membrane to the soluble fraction (Johnson et al., 1990). Significantly, the modification of v-Src with 13-oxamyristate (12-methoxydodecanoic acid) or 6-oxamyristate leads to a substantial redistribution of the protein to the cytosol (Johnson et al., 1990). In unpublished studies, we have found that 12-methoxydodecanoic acid can also be incorporated into Lck and Lyn, but does not displace these from the membrane. These compounds are interesting because of their selectivity, but have not been examined extensively in hematopoietic cells for their abilities to alter the signaling properties of SFKs.

The S-acylation of Fyn in intact cells is more heterogeneous than N-acylation with small amounts of palmitoleate, oleate and stearate being present along with a preponderance of palmitate (Liang et al., 2004). Thus, the palmitoylation reaction may be less rigorous in terms of substrate selection than myristoylation. In fact, the incubation of cells with exogenous, polyunsaturated fatty acids leads to their incorporation into Fyn in place of palmitate (but not myristate) resulting in the displacement of Fyn, not from the membrane, but from lipid rafts (Webb et al., 2000; Liang et al., 2001). This results in defective signaling through the TCR. In a related approach, Lck also can be displaced from lipid rafts in T cells by treatment with 13-oxypalmitic acid (13-OP) (11), a less-hydrophobic, heteroatom-substituted derivative of palmitic acid (Hawash et al., 2002a). Treatment of cells with 13-OP abolishes the association of Lck with the raft-associated protein, CD48, but not with the transmembrane, but nonraft protein, CD4 and also inhibits TCR signaling. These results support the idea that SFKs must not only be localized to the membrane but also to specific membrane subdomains to function efficiently in T-cell signaling. This function can be interdicted by fatty acid analogs that do not satisfy the tight acyl chain packing required for the localization of proteins to membrane rafts.

Interdiction through binding partners

Since SFKs are activated by tyrosine phosphorylation, it seems obvious that the main modulators of negative regulation of SFKs are protein tyrosine phosphatases (PTPs). The immediate complication to this simplistic view is that SFKs also are inhibited by tyrosine phosphorylation and this duality sets the stage for a much more complicated yin and yang model of regulation. Specifically, it is becoming increasingly appreciated that palmitoylated adaptor proteins thought to be localized in lipid rafts play defining roles in the regulation of SFKs. Although mutational studies early on demonstrated the importance of the binding modules (SH2 and SH3 domains) in regulating SFK function, in many cases the critical binding partners were not understood.

As the SFK Lck is one of the best-studied SFKs, we will use it as a model in discussing the intricacies that underlie the regulation of SFKs. However, evidence exists that the adaptor proteins mediating Lck regulation are expressed or have counterparts in several hematopoietic cell types. For example, the Csk-binding protein PAG/Cbp is ubiquitously expressed and while the adaptor protein LAT functions downstream of the TCR, FcRI, and the GPVI receptor in platelets (Pasquet et al., 1999; Zhang et al., 1999; Saitoh et al., 2000), a functional equivalent of LAT, LAB/NTAL, functions downstream of the BCR, FcRI and FcRI (Brdika et al., 2002). The newly discovered T-cell adaptor protein, LIME, in addition to being expressed in peripheral T cells also is expressed in NK T cells and macrophage cell lines (Brdiková et al., 2003; Hur et al., 2003).

As described elsewhere in this volume, Csk and its family member Chk are the tyrosine kinases responsible for phosphorylating SFKs at their inhibitory site (Veillette et al., 2002). Csk however, is primarily cytosolic and the mechanism underlying its recruitment to the plasma membrane, although presumed to involve its SH2 and/or SH3 domains remained largely elusive prior to the discovery of the transmembrane Csk-binding protein PAG/Cbp (Brdika et al., 2000; Kawabuchi et al., 2000). The palmitoylated PAG/Cbp is localized in membrane lipid rafts in resting T cells where it is constitutively tyrosine phosphorylated by SFKs and binds Csk through the Csk SH2 domain (Brdika et al., 2000; Kawabuchi et al., 2000; Yasuda et al., 2002). Csk in turn is bound to the PTP PEP, which is thought to dephosphorylate the active site of SFKs (Cloutier and Veillette, 1996). Thus, PAG/Cbp recruitment of the Csk/PEP complex provides a mechanism for dephosphorylating the active site, while phosphorylating the inhibitory site of SFKs. The immediate obvious consequence of the localization of Csk in lipid rafts is that raft resident SFKs would be phosphorylated on their inhibitory tyrosine site and inactive. Following receptor binding, PAG/Cbp is dephosphorylated and Csk returns to the cytosol (Brdika et al., 2000). Both the transmembrane PTP CD45 (Davidson et al., 2003) and the SH2 domain-containing PTP Shp-2 (Zhang et al., 2004) have been implicated in the dephosphorylation of PAG/Cbp. Although this model explains the finding that Lck in lipid rafts is relatively inactive (Kabouridis, 2003 and see below), it is difficult to reconcile the model with the presence of active Lyn in lipid rafts in resting RBL cells (Young et al., 2003).

Along with the signaling regulator PAG/Cbp, two recently identified binding partners for Lck, LAT (linker of activated T cells) and LIME (Lck interacting molecule/membrane protein) are palmitoylated and localized to lipid rafts. Both are tyrosine phosphorylated by SFKs following TCR and coreceptor engagement (Brdiková et al., 2003; Hur et al., 2003). LIME binds Lck through the Lck SH2 domain (Brdiková et al., 2003; Hur et al., 2003). Although LAT was recently shown to co-precipitate with Lck (Kabouridis, 2003), it is not yet clear whether this is a direct interaction. Importantly, lipid raft-associated Lck in resting cells was found to be in the closed, inactive conformation and the presence of LAT was necessary to maintain this inactive conformation (Kabouridis, 2003), adding LAT to the list of negative regulators of Lck. This finding is consistent with the constitutive presence of Csk in lipid rafts in nonactivated cells. Raft-resident LAT was found to interact with the open active form of Lck. This finding led to a model where the active form of Lck is interdicted by LAT in lipid rafts leading to the rapid inactivation of Lck by colocalized Csk (Kabouridis, 2003).

Raft-resident LIME binds both Lck and Csk following receptor stimulation. LIME also associates with the open, active form of Lck, but this association occurs following engagement of the TCR coreceptor CD4 (Brdiková et al., 2003). Additionally, it was found that LIME-associated Lck was highly phosphorylated on the C-terminal inhibitory site (Brdiková et al., 2003). The simultaneous association of LIME with Lck and Csk led Brdiková et al. (2003) to propose the following model: following crosslinking of CD4, active Lck phosphorylates LIME generating binding sites for itself and Csk. Csk phosphorylates Lck on its inhibitory site; however, this does not result in inhibition since the SH2 domain of Lck that engages the inhibitory site phosphotyrosine is unavailable due to its interaction with a phosphotyrosine residue on LIME. A most exciting potential consequence of generating an unengaged inhibitory site phosphotyrosine in a SFK is that this sets up a docking site for other SH2 domain-containing signaling proteins (Brdiková et al., 2003 and B Schraven, personal communication). As discussed by Brdiková et al. (2003), this could explain the finding that Lck is activated outside of lipid rafts, but rapidly translocates into lipid rafts where it is needed for the activation of Fyn. Perhaps the presence of the free phosphotyrosine at the inhibitory site in Lck provides a binding site for the SH2 domain of Fyn, resulting in Fyn activation. A model of the interaction of SFKs and their binding partners and regulators is shown in Figure 2.

Figure 2.

A model of the interactions of SFKs with each other and raft-resident binding partners. The model highlights the potential for an interaction between the phosphotyrosine residue at the inhibitory site and the SH2 domain of other Src kinases or signaling molecules. This interaction results when the inhibitory tyrosine is phosphorylated in an SFK whose SH2 domain is bound to a binding partner. (a) The raft-resident adaptor protein LIME is phosphorylated by Lck following T-cell activation. Tyrosine phosphorylated LIME binds both Csk and Lck enabling Csk to phosphorylate the inhibitory site on Lck. (b) Since the Lck SH2 domain is bound to LIME, the inhibitory site phosphotyrosine cannot bind, thereby providing a binding site for another protein with an SH2 domain such as Fyn. (c) Active Fyn (or active Lck) phosphorylates raft-resident PAG/Cbp providing a binding site for the SH2 domain of Csk enabling Csk to phosphorylate and inactivate molecules of Lck and Fyn whose SH2 domains are not engaged. Closed circles are tyrosine residues phosphorylated by SFKs, whereas open circles are tyrosine residues phosphorylated by Csk

Full figure and legend (66K)

The recent reports of lipid raft-resident binding partners for SFKs represent an emerging story of microdomain-localized regulation that appears to be complex. Unraveling the functional consequences of these associations will be important as will be determining how SFKs that are localized outside of lipid rafts are regulated. The picture at present is more tantalizing than clear and further studies are eagerly anticipated.

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Acknowledgements

Work in the author's laboratories is supported by grants NIH CA37372 (RLG) and NIH GM48099 (MLH). M Handley is supported by NIH Training Grant T32CA09634 awarded to the Purdue Cancer Center.