The tyrosine kinase network regulating mast cell activation

Abstract

Mast cell mediator release represents a pivotal event in the initiation of inflammatory reactions associated with allergic disorders. These responses follow antigen-mediated aggregation of immunoglobulin E (IgE)-occupied high-affinity receptors for IgE (Fc epsilon RI) on the mast cell surface, a response which can be further enhanced following stem cell factor-induced ligation of the mast cell growth factor receptor KIT (CD117). Activation of tyrosine kinases is central to the ability of both Fc epsilon RI and KIT to transmit downstream signaling events required for the regulation of mast cell activation. Whereas KIT possesses inherent tyrosine kinase activity, Fc epsilon RI requires the recruitment of Src family tyrosine kinases and Syk to control the early receptor-proximal signaling events. The signaling pathways propagated by these tyrosine kinases can be further upregulated by the Tec kinase Bruton's tyrosine kinase and downregulated by the actions of the tyrosine Src homology 2 domain-containing phosphatase 1 (SHP-1) and SHP-2. In this review, we discuss the regulation and role of specific members of this tyrosine kinase network in KIT and Fc epsilon RI-mediated mast cell activation.

Fig. 1. Signaling pathways leading from activated KIT and aggregated FcεRI to mast cell responses

Antigen-induced aggregation of IgE-occupied FcεRI induces activation of the Src family tyrosine kinase, Lyn, whereas SCF-induced KIT dimerization induces activation of its intrinsic KIT kinase activation. Phosphorylation of tyrosine residues within the receptor chains thus allows recruitment of SH2 domain-containing signaling molecules. In the case of FcεRI, Syk is recruited via ITAMs contained in the γ chain-cytoplasmic domains. Resulting activation of Syk, following its phosphorylation, leads to consequential phosphorylation of the transmembrane adapter molecules LAT1 and LAT2 (NTAL/LAB). Upon phosphorylation, these proteins serve as scaffolds for multimolecular signaling complexes comprising various cytosolic adaptor molecules such as Gads, Grb2, SLP76, and SHC, GTP exchangers including Sos and Vav1 and the signaling enzymes PLC_γ1 and PLCγ₂. PLCγ catalyzes the hydrolysis of PtdIns₂ to yield diacylglycerol (DAG) and IP₃, which, respectively, result in the activation of PKC and the liberation of intracellular calcium. Following depletion of the intracellular calcium stores, the calcium signal is maintained by store operated calcium entry (not depicted). These signals lead to mast cell degranulation and eicosanoid generation and also contribute to activation of transcription factors required for cytokine and chemokine production. In parallel to this pathway, PI3K is activated following binding to Gab2 upon the phosphorylation of this cytosolic adapter molecule by Fyn and/or Syk, phosphorylation of the p85α adapter subunit of PI3K, and activation of the catalytic subunit by small GTP-binding proteins. In the case of KIT, the p85α subunit directly binds to the phosphorylated molecule. The subsequent formation of membrane associated PtdInsP₃ results in the recruitment of PH domain-containing signaling molecules such as Btk, PLD, and potentially others. PI3K-regulated pathways serve to enhance/maintain LAT/PLC_γ1-regulated degranulation and, as depicted for KIT, regulate mast cell growth, differentiation, survival, migration, adhesion, and cytokine production. KIT- and FcεRI-mediated activation of the Ras-Raf-MAPK pathway following Sos- and Vav-regulated GDP-GTP exchange of Ras also contributes to these processes. The MAPK ERK1/2 also regulates PLA₂ activation, which leads to the liberation of arachidonic acid for the generation of eicosanoids (not depicted). The role of LAT2 in mast cell activation is still enigmatic; however, it has been proposed to both upregulate and downregulate antigen-mediated responses. It does appear to be required for the ability of KIT to enhance FcεRI-dependent degranulation. Due to the complexity of the signaling cascades depicted, some of the intermediary steps involved in these processes could not be illustrated in this figure. For further details, readers are referred to other recent review articles (6,7,11).

Fig. 2. Basic structures of the major tyrosine kinases involved in mast cell activation

The major tyrosine phosphorylation sites are represented by red circles. The tyrosine numbers designated for KIT, Lyn, Fyn, Btk, and Itk are based on the human sequence and for Syk on the rat sequence. The major signaling molecules recruited to specific phosphorylated tyrosines are depicted for KIT. PR in the Tec kinases structure represents a proline-rich region. A and B in the Syk structure shows the interdomain regions A and B of this kinase.

Fig. 3. Regulation of Src PTK recruitment to FcεRI

The equilibrium of active and inactive Src PTKs, like Lyn, normally favors their inactive or closed conformation (as depicted on the left). This is mediated through the interaction a negative regulatory tyrosine (Y) at the COOH-terminus of Src PTKs (Y⁵⁰⁸ for Lyn) with its own SH2 domain. This balance is regulated by CD45 and Csk. In resting cells, Csk phosphorylates this site maintaining the closed conformation. FcεRI stimulation shifts the balance to CD45, which dephosphorylates Y⁵⁰⁸ allowing the SH2 domain to become available and bind this receptor. Once open, the phosphorylation of the activation loop tyrosine (Y³⁹⁷) increases, enhancing activity and stabilizing the open/active conformation. Fyn recruitment seems to function similarly although where this interaction might occur is not yet defined.

Fig. 4. Cbp/CSK-mediated regulation of Fyn activity

In resting mast cells, active Lyn kinase phosphorylates the adapter Cbp at Y³¹⁴ at low levels. Phosphorylation of this site causes the binding and membrane localization of CSK, which is normally localized in the cytoplasm. CSK can then target the COOH-terminal negative regulatory tyrosine (Y) residue found in Src PTKs. In Fyn, Y⁵²⁵ is a target for CSK activity leading to Fyn inactivation by inducing a closed conformation via intramolecular interaction with its own SH2 domain. The low level of Cbp phosphorylation maintains the equilibrium between active and inactive Src PTKs in resting cells. Upon FcεRI stimulation, the balance shifts towards increased activation of Src PTKs, likely through CD45. This new equilibrium is governed by a Lyn-dependent increase in Cbp phosphorylation and membrane CSK recruitment, which controls the activity of Fyn by inactivation as described above. Thus, the absence of Lyn leads to a loss in control of Fyn activity and mast cells are hyperresponsive (63, 85).

Fig. 5. Basic structures of the tyrosine kinase, CSK, and the tyrosine phosphatases SHP1, SHP2, and CD45

Several splice variants of various lengths of CD45 have been described as depicted by A, B, and C in the figure.