Lyn, a src-like tyrosine kinase

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J. Bioclrcv~~. (‘e/l Bd. Vol. 29. No. 3. PP. 397-400. 1997 , 1097 Elscvw Saence Ltd. All rights reserved

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IN FOCUS

Lyn, a svc-like Tyrosine Kinase MARGARET

L. HIBBS,

ASHLEY

R. DUNN*

Ludwig Institute for Cancer Research, Melbourne Tumour Biology Branch, P.O. BO.Y2008, Ro!d Melbourne Hospital, Victoria 3050, Australia LJY~ is a member of the WC family of non-receptor protein tyrosine kinases that is predominantly expressed in haematopoietic tissues. Like all members of the SIT family, fyn is thought to participate in signal transduction from cell surface receptors that lack intrinsic tyrosine kinase activity. It is associated with a number of cell surface receptors including the B cell antigen receptor and FccRI. Lyn deficient mice develop autoimmune disease characterised by autoantibodies in serum and the deposition of immune complexes in the kidney, a pathology reminiscent of systemic lupus erythematosus. Lyn deficient mice also have impaired signalling involving FURI in mast cells, resulting in defective allergic responses. !(“I 1997 Elsevier Science Ltd ht. J. Bioclwm.

Cdl Bid.

(1997) 29, 397-400

INTRODUCTION

Protein tyrosine kinases (PTKs) are categorised as either: (1) receptor PTKs which contain extracellular, ligand binding domains and transmembrane domains; or (2) cytoplasmic non-receptor PTKs. PTKs function in transducing signals from the extracellular environment to the nucleus to induce cellular changes such as proliferation, differentiation or apoptosis. Nonreceptor PTKs. which lack extracellular domains. participate in these functions through association with the cytoplasmic domain of non-catalytic cell surface receptors. There are at least eight different subclasses of non-receptor PTKs and the SYCfamily, which consists of src, ,fjx. J’PS, Iyrz, Ick, hck, jir, blk and yrk, is amongst the best characterised. The src family kinase, Iyn, was first identified in 1987 as a gene with high homology to other members of the src family (Yamanashi et al., 1987). While all members of the src family are thought to have arisen by repetitive duplication of a primordial src-like gene, hck and lyn are believed to have evolved subsequently from a more recent common ancestor. Studies on the expression pattern of Iyn have shown that it is *To whom all correspondence should be addressed. Received 20 May 1996: accepted 3 September 1996. 397

found predominantly in haematopoietic cells (Yi et al., 1991; Stanley et ul., 1991; Yamanashi et al., 1989). STRUCTURE

The human low cDNA was isolated from a placental library under conditions of reduced stringency using a v-yes-specific probe, and was found to be highly homologous to other members of the src-family (Yamanashi et al., 1987). The structure of Liz is shown in Fig. 1. For a more comprehensive review on the structure of src family PTKs, refer to SupertiFurga and Courtneidge, 1995. Lyn exists as two distinct isoforms, designated ~56’“” and p53”“, which are derived from alternatively spliced mRNAs (Yi et al., 1991; Stanley P/ rrf., 1991). The protein encoded by ~56’~” contains 5 12 amino acids, while ~53’“” contains 491 amino acids. The amino-terminus of I>x contains a consensus sequence for attachment of the fatty acid myristate. During translation, the aminoterminal methionine is removed and myristate is attached covalently to the adjacent glycine. This modification is required for association of 1~11 with the plasma membrane. The amino-terminal attachment domain is followed by a “unique”

3YX

Margaret

L. Hibbs

domain of approximately 50 amino acids that is the least conserved region between ,YI’(’family members, and is thought to be important for specifying the interaction of /~v7 (or indeed any .src~-family PTK) with upstream target proteins. Intriguingly. the only differences between the /IX isoforms are in the unique domain; ~56”” contains an additional 21 amino acids. While both /yn isoforms are co-expressed and have been found to be associated with the same cell surface receptor complex, it is possible that they mediate binding to different cell surface receptors through their unique domain sequences, or that they have different affinities for the same receptor complex. Following the unique domain are the src-homology-3 (SH3) and src-homology-2 (SH2) domains comprised of approximately 60 and 100 amino acids. respectively. These domains are important in protein-protein interactions; the SH3 domain in binding proline-rich motifs and the SH2 domain in recognising phosphotyrosine-containing proteins. The carboxy-terminal half

WC family ubiquitous

Structure:

NH2

Ashley

tyrosine

kinases fgr, Ick, b/k

lyn, hck predominantly haematopoietic

KINASE *J-e

R. Dunn

of IJJ~contains the catalytic or kinase domain and is the most conserved region between .SY~ family members. A characteristic feature of the catalytic domain is the presence of a tyrosine residue (Tyr-397, the autophosphorylation site) which becomes phosphorylated during enzyme activation (Fig. I). At the carboxy-terminus of lyn is a region of approximately 20 amino acids. referred to as the regulatory domain. which includes a conserved tyrosine residue (Tyr-508) the phosphorylation of which negatively regulates Ilw tyrosine kinase activity. Current models suggest that phosphorylated Tyr-508 interacts with its own SH2 and SH3 domains, thereby assuming a conformation which reduces kinase activity (Fig. I). Dephosphorylation of lyn at Tyr-508 results in a change in conformation which promotes enzyme autophosphorylation and hence an increase in kinase activity (Fig. 1). LJw activity is in part regulated by the PTK csk (Cooper and Howell, 1993). All .YI‘(’PTKs are highly conserved at the genomic level, suggesting that they have arisen

of protein

src, yes, fyn, yrk Expression:

and

DOMAIN

haematopoietic

COOH

“ai+. 00

csk and other

Fig. 1. Structure, expression pattern and regulation of .FW family PTKs. The nine characterised members of the src family have distinctive expression patterns; SW, yes, [rn and yrk have a wide-spread distribution whereas the other members are found either predominately or exclusively in haematopoietic tissues. All members of the src family are structurally homologous. and their activity is believed to be regulated by a common mechanism. Src PTKs are inactivated by phosphorylation of their terminally located regulatory tyrosine; the phosphotyrosine interacts with the enzymes SH2 and SH3 domains. Activation is associated with dephosphorylation of the regulatory tyrosinc and autophosphorylation.

Lyn,

a src-like

by duplication of a prototypic src-like gene. While the overall structure of the mouse lyn gene is similar to that of other src family members, a unique feature is the presence of an internal duplication between intron 1 and 10 (the gene is comprised of 13 exons) (Hibbs et al., 1995a). While nucleotide sequence analysis and Southern blotting studies have shown that the duplication is a recent evolutionary event, these duplicated sequences are not expressed (Hibbs et al., 1995a). BIOLOGICAL

FUNCTION

A major clue in unravelling the function of 1~~1as a signal transduction molecule was the demonstration that Iyn is physically associated with a number of haematopoietic cell surface receptors including the B cell antigen receptor (BCR), the high affinity FctRI complex, CD40, FcyRI, the lipopolysaccharide receptor, and several cytokine receptors. More recent biochemical studies suggest that fyn plays a dominant role in signal transduction mediated by the BCR (reviewed in Pleiman et al., 1994). The BCR complex consists of an antigen-binding membrane immunoglobulin non-covalently associated with disulphidelinked heterodimers of Ig-cc and Ig-p subunits. LJW is associated with the cytoplasmic domain of the lg-z subunit and is rapidly activated on BCR stimulation; it phosphorylates critical tyrosine residues that are present in highly conserved motifs (ITAMs) within the cytoplasmic domain of Iga and Igfi molecules, creating binding sites for SH2-containing molecules thought to be involved in the BCR signal transduction cascade. Lyn most likely regulates the activity of another cytosolic tyrosine kinase, sq#c, that is also rapidly activated following BCR cross-linking. The generation of /J~Zdeficient chicken B cell lines has revealed perturbed biochemical responses following BCR cross-linking such as delayed and slow calcium mobilisation, and profoundly reduced phosphorylation and activation of syk (Takata et al., 1994). Detailed biochemical studies have also begun to unravel the signalling molecules coupled to the Fct Rl complex, and implicate lyn as a major component (reviewed in Ravetch, 1994). Crosslinking of the FURI complex, which is made up of a ligand binding c( subunit, a /? subunit and homodimeric ;’ subunits, results in the rapid phosphorylation and activation of lyn and syk.

tyrosine

kinase

Tyrosine residues within ITAMs of both the /I and y subunits of FcERI are targets for phosphorylation; these phosphotyrosines presumably recruit other molecules involved in the signal transduction cascade. While these biochemical studies provide compelling evidence for the importance of lyn as a signal transduction component of haematopoietic cells, the physiological relevance of lyn in haematopoietic signalling pathways was only demonstrated recently by the generation of 1~11 deficient mice. Ourselves (Hibbs et al., 1995b) and others (Nishizumi et al., 1995) have demonstrated that /yn is an indispensable component of the BCR complex. Lyn deficient mice have an impairment of immunoglobulin-mediated signalling manifested by reduced numbers of peripheral B cells, failure to mount effective immune responses against T-independent and T-dependent antigens, elevated levels of serum IgM and most strikingly, the development of a severe autoimmune disease. Studies from our laboratory have also shown that Iyn is a crucial component of the FctRI complex and is indispensable for mast cell-mediated allergic responses (Hibbs et al., 1995b). CLINICAL

IMPLICATIONS

Our recent studies have shown that IJsn deficient mice develop autoimmune disease characterised by the appearance of anti-nuclear antibodies in serum, and pathogenic immune complexes in the kidney (Hibbs et al., 1995b). The glomerulonephritis and pancytopenia seen in lyn deficient mice bear many similarities to the renal and hematologic pathology associated with the human disorder, systemic lupus erythematosus (SLE), a disease characterised by the production of autoantibodies and immune complex deposition. While much of the pathology associated with SLE, such as polyarthritis and vasculitis, are not seen in Ivn deficient mice, it is possible that a subset of patients with SLE may carry mutations in their lyn gene. Such an hypothesis can be tested by examining the kinase activity of IJX in peripheral blood leukocytes from SLE patients and subsequently by sequencing the 1)x gene. In addition to the importance of Iyn in B cell signalling, our recent studies have also shown that l~rzplays a critical role in mast cell function and specifically in signalling from FccRI, the receptor involved in the allergic response (Hibbs et ul., 1995b). LJW deficient mice are unable to

400

Margaret

L. Hihbs

mediate passive cutaneous anaphylaxis. a model allergic reaction, suggesting that /JYTwould be an excellent target for allergy therapy. While early studies have attempted to target the Fcf RI itself. inhibitors of 1~~~~ kinase activity would also provide a rational alternative for the treatment or prevention of allergic reactions. REFERENCES Cooper J. A. and Howell B. (1993) The when and how of Src regulation. Cc// 73, IO.51 1054. Hibbs M. L., Stanley E.. Maglitto R. and Dunn A. R. (1995a) Identification of a duplication of the mouse LIX gene. G’cnr 156, 175-181. Hibbs M. L.. Tarlinton D. M., Armes J.. Grail D., Hodgson G., Maglitto R.. Stacker S. A. and Dumi A. R. (1995b) Multiple defects in the immune system of /w-deficient mtcc. culminating in autoimmune disease. Co/l 83, 301 -311. Nishirumi H., Taniuchi 1.. Yamanashi Y.. Kitamura D., IIic D.. Mori S.. Watanabe T. and Yamamoto T. (1995) Impaired proliferation of peripheral B cells and indication of autoimmune disease in LJwdeficient mice. hwn~nir~ 3, 5499560. Pleiman C. M., D’Ambrosio D. and Cambier J. C. (1994) The B-cell antigen receptor complex: structure and signal transduction. hnmunol. T&I, 15. 393-399.

and Ashley

R. I>unn

Ravetch J. V. (1994) Fc receptor<: rubot- redux. Cc,// 78. 553 560. Stanley E.. Ralph S.. McEwen S.. Boulct 1.. Holtrman D. A.. Lock P. and Dunn .A. R. (1991) .Altcrnativcly