Novartis Foundation Symposium Edited by Ryo Sato, Gregory R. Bock, Kate Widdows Copyright 0 1992 by Ciba Foundation
Protein tyrosine kinases belonging to the src family Kumao Toyoshima*, Yuji Yamanashit, Kazushi Inoue*, Kentaro Sembat, Tadashi Yamamotof and Tetsu Akiyama* *Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565 and tlnstitute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108, Japan
Abstract. There are nine non-receptor-type protein tyrosine kinases that show a high level of similarity in their primary structures and in the structures of their functional domains. Together, they are called the src family. They seem to have common sites specific for oncogenic activation. Recent findings suggest that the kinases are closely associated with cell surface molecules and that they mediate extracellular signals through the activation of their tyrosine kinase activity. They appear to act more on the differentiated phenotype than in haemopoietic cell proliferation. Possible functions of the products of the Ick,fyn, lyn and fgr genes in lymphocytes and rnonocytes are discussed. I992 Interactions among cell signalling systems. Wiley, Chichester (Ciba Foundation Symposium 164) p 240-253
Protein tyrosine kinases (PTKs) are divided into two major categories-the receptor type and the non-receptor type. Kinases in the former category, represented by the product of the erbB/EGF receptor gene or the fms/CSF (colony-stimulatingfactor)-1 receptor gene, are encoded by several gene families. Similarly, the non-receptor tyrosine kinases are also encoded by at least four families; namely src, abl, fps/fes and src-kinase. The last enzyme is a novel ~ ~ product , of the PTK responsible for phosphorylation of Tyr-527 of P ~ O C - ~the c-src gene (Okada 8c Nakagawa 1989). PTKs in each family show structural similarity, as well as sharing genomic structure (such as that of the splicing junctions of the coding region; Toyoshima et a1 1987). However, the structure of the regions that regulate gene expression are specific to each gene. These similarities and specificities suggest that the members have common mechanisms for regulation of their enzymic activities, but have expression patterns that differ, possibly according to their particular function (Toyoshima et a1 1990). The biological significance of receptor-type PTKs has been widely discussed by many research groups, but the functions of the non-receptor type PTKs in normal states have been clarified only recently. The src gene, the representative of the src family, was known to be expressed in neuronal cells (Sudoh 1988), 240
src family protein tyrosine kinases
24 1
possibly playing a role in differentiation (Bjelfman et a1 1990), though the pathway of the signal is still unknown. The first evidence for a link between an external signal and a non-receptor type PTK was the association of p56Ick with CD4/CD8 antigen in T cells (Rudd et a1 1988, Veillete et a1 1988). Here, we describe significant aspects of the structure of the src family and discuss possible functions of some members of this family in haemopoietic systems. Structural similarity in the src family kinases Nine members of the non-receptor type PTK family are reported to have highly '~ 1). These kinases have conserved structure, typified by that of P ~ O C - ~ (Fig. molecular masses around 55 to 60 kDa and have a glycine residue at position 2, which is myristoylated after removal of the N-terminal methionine. This myristic acid and several amino acids next to Gly-2 are believed to be involved in the localization of the enzyme in the plasma membrane (Shulz et a1 1985). In fact, PTKs of this family are known to associate with membrane fractions; this is in contrast to the cytosolic or nuclear localization of other non-receptor type PTKs, such as Fps and Abl.
I
I GK
Y416
Y529
I
533
60.0
543
60.0
IG
I
I GK
Y
Y
I
529
59.5
IG
1
I GK
Y
Y
l
537
60.0
rG
I
I GK
Y
Y
1
512
50.6
IG
I
I GK
Y
Y
I
509
56.0
16
I
I GK
Y
Y
I
505
55.5
IG
I
I GK
Y
Y
]
499
55
?
?
FIG. 1. Schematic structures of six non-receptor-type protein tyrosine kinases. M, myristic acid; P, phosphate; G, glycine; K, lysine; Y, tyrosine. Numbers (right) indicate the number of amino acids and molecular mass in kDa Cfar right) of each kinase.
Toyoshima et al
242
EGFR p98f-fPs
SH2
1
p145c'ab1
P60c-src p45 gag-crk
PLC
GAP
527
UNIQUE SH3 gag
SH2
SH2
SH2 #H3
SH2 HSH
KINASE
R
b
SH2
a-SPECTRIN FIG. 2. A model of the functional domains of the src family tyrosine kinases. Additional proteins that have regions homologous to the modulatory domain are illustrated. EGFR, epidermal growth factor receptor; PLC, phospholipase C-y,; GAP, GTPase-activating protein.
Following the N-terminal sequence is a region of about 50 to 80 amino acids that is unique to each member-almost no homology is evident in this region. The difference in molecular size of the kinases is mostly due to the variation in the number of amino acids in this region. The region's function is still not clear, but it is postulated to play a role in association of the kinase with a surface molecule which is able to receive extracellular signals. The next region of the sequence, about 150 amino acids, has moderate similarity among members of the src family, and includes src-homologous (SH) regions. These SH regions were originally found as conserved regions in other non-receptor-type PTKs such as the abl and f p s gene products. This region is also conserved in a variety of cytoplasmic molecules that are thought to interact with PTKs (Fig. 2), and is considered to be the site that interacts with cytoplasmic proteins to modulate signals. The C-terminal half of the molecule is a highly conserved region; sequence identity is about 80 to 90%. This region includes the catalytic domain, which phosphorylates tyrosine residues of target proteins, and the C-terminal regulatory domain, which down-regulates the catalytic activity when the tyrosine at the most C-terminal position is phosphorylated. Similarity and dissimilarity of genomic structures The genomic organization of the coding regions of c-src is identical in the chicken and human (Takeya & Hanafusa 1983, Anderson et a1 1985). When the human c-fgr clone was analysed, the splicing junctions of exons 4 to 12 were
src family protein tyrosine kinases
243
found to coincide with those of the c-src gene. Similar coincidences of splicing junctions were also found in the c-yesgene (Nishizawa et al1985, 1986). Possible splicing junctions found in other members of the src family were also in the same locations as those of the c-src gene, except in the unique region. In contrast, splicing junctions in the c-erbB2 gene were completely different from those of the src family, even within the catalytic domain, suggesting that the nine members of the src family arose by duplication of a preformed ancestral gene. In contrast, regulatory elements located upstream of the src family genes are specific to each member of the family. This difference of regulatory elements may explain the unique pattern of each expression of src family genes in different tissues and organs. Oncogenic activation of the sre family genes The c-src gene, integrated into a retroviral genome, acquires cell-transforming capacity only after mutation. v-src, v-yes and v-fgr were found as oncogenes of acutely oncogenic retroviruses. All of these, including S1 and S2, new isolates containing activated c-src (Ikawa 1986), have lost a sequence encoding the Cterminal portion of their regulatory domains through recombination with viral or other cellular sequences. Thus, they have invariably lost a tyrosine residue at the most C-terminal position (corresponding to Tyr-527 of the c-src gene product). Substitution of phenylalanine for Tyr-527 also activated tyrosine kinase activity and added transforming capacity to p60c-src.In addition, substitution of an amino acid residue in the modulatory domain (Tyr-90 to Phe; Tyr-92 to Phe; Arg-95 to Glu, Lys or Trp) or in the kinase domain (Thr-338 to Ile; Glu-378 to Gly; Ile-441 to Phe), or deletions including part or almost all of the modulatory domain also confer transforming activity on p60c-src.These mutations are also known to activate the tyrosine kinase activity of the enzyme. Activation of other members of the family has not been tested extensively; deletion of C-terminal sequences including the tyrosine residue corresponding to Tyr-527 of p60c-src,or substitution of the same tyrosine by phenylalanine, activates both tyrosine kinase and transforming activities in c-Yes, c-Fgr, Fyn, Lck and Hck. Deletions in the modulatory domain and substitution of an amino acid in the kinase domain in Fyn are also reported to activate transforming capacity (Semba et a1 1990). These mutations and substitutions are summarized by Cooper (1990) and Semba & Toyoshima (1990). A precise analysis of mutations within the modulatory domain of p6WCwas published recently by Hirai & Varmus (1990). lck and fyn expression in T cells
ick was identified as a gene encoding a novel tyrosine kinase, p56Ick,that is abundantly expressed in mouse LSTRA (T cell lymphoma) cells (Marth et a1 1985, Voronova & Sefton 1986). Its human counterpart was soon found to be
244
Toyoshima et al
expressed in T lymphocytes. The dramatic decrease of p56/ckin T cells during their activation suggests that it plays an important role in this process (Marth et a1 1987). The protein appears to be functionally and physically associated with CD4 or CD8 in normal T cells, and cross-linking of CD4 activates the protein tyrosine kinase activity of p56lck(Veillette et a1 1988). Activation of T cells occurs when the T cell receptor is stimulated by a specific antigen and the CD4 antigen interacts with a class I1 major histocompatibility complex (MHC) molecule. The association of CD4 with p56ICk is critical in this process (Glaichenhaus et a1 1991). High expression of fyn was observed in T cells of mice with the autosoma1 recessive mutation Ipr (lymphoproliferation) or gld (generalized lymphoproliferative disease). These homozygous mice with autoimmune disease have polyclonal proliferation of CD4-/CD8- T cells and lymph node enlargement (Katagiri et a1 1989). Expression offyn is low in T cells of normal mice that have the same genetic background except for the lpr or gld gene. p56lckis expressed at similar levels in diseased and normal mice. The expression offyn in negatively selected CD4- K D 8 - T cells was increased 10-fold about two hours after stimulation by treatment with anti-CD3q concanavalin A, 12-O-tetradecanoylphorbol-13-acetate(TPA) or A23 187 (a calcium ionophore), while lck expression remained constant. In addition, p59fYn and CD3 were coprecipitated by anti-CD3. Thus, p56lckassociated with CD4/CD8 aids in the recognition of MHC molecules and p59fYn associated with CD3 is involved in the response to growth-stimulating treatment. CD45, a common lymphocyte antigen that has phosphotryosine phosphatase activity, is thought to activate p56ICktyrosine at position 505 (Ostergaard et a1 1989). Thus, at least three components may act in the regulation of T cell growth in response to a stimulus from outside the cell. Association of p56'Yn with the B cell antigen receptor complex Iyn is preferentially expressed in B cells in the lymphoid system (Yamanashi et a1 1989). p56'Yn is not readily precipitated as an immune complex by known antibodies to lymphocyte surface molecules. However, mild treatment with digitonin of WEHI-231 cells, a mouse B cell line, allowed us to co-immunoprecipitate p56bn with surface membrane-bound IgM. This loose association between p56IYn and the IgM can be expected because B cell antigen receptors have only a few amino acids in their cytoplasmic regions (Rogers et a1 1980). WEHI-231 cells produce two types of p chain-a membrane form (pM) and a soluble form (pS). pM was co-immunoprecipitatedwith p56/yfl by anti-Lyn, and IgM-associated molecules of 30 to 40 kDa were co-immunoprecipitated with p56bn by anti-IgM (Yamanashi et a1 1991). Cross-linkage of membrane IgM up-regulates the tyrosine kinase activity of ~56~Y",and phosphorylation at tyrosine residues of at least 10 molecules increases within
src family protein tyrosine kinases
245
0 0
om*. 0.0
Ick fY n
hCk
fgr
mast cell
lYn blk
src fgr l Yn
plasma cell macrophage stem cell
FIG. 3. cells.
Expression of cytoplasmic tyrosine kinases of the src family in haemopoietic
2-10 min after cross-linkage. As a result of this treatment p56'Yn was downregulated instantly, reaching a minimum level about 30 min after stimulation, whereas p53/Yn,which was also associated with IgM, was not down-regulated. These results, together with the recent finding that p56'vn and membrane IgD can be co-immunoprecipitated (Y. Yamanashi, T. Yamamoto & K. Toyoshima, unpublished work), suggest that p56bn plays an important role in antigendependent B cell differentiation. Although there is no evidence, CD45 and p5Sb" (Dymecki et a1 1990) may also play an important role in B cell proliferation and differentiation (Fig. 3). The connection between an 80 kDa tyrosine kinase described by Campbell & Sefton (1990) and p56[Yn is unclear at present.
246
Toyoshima et al
Possible functions of pSsfgr in monocytes
p58c-jgris known to be expressed in B cells infected with Epstein-Barr virus. Recently, we reported that the enzyme is specifically expressed in peripheral monocytes, granulocytes and natural killer cells. When HL-60, a promyelocytic cell line, was treated with retinoic acid or TPA to induce myelomonocytic differentiation, p5SC-fgraccumulated in differentiated cells (Inoue et a1 1990, Kawakami et a1 1988), suggesting that ~58~-fg' is more closely associated with differentiation than with proliferation. The c-fgr gene and a mutant encoding phenylalanine instead of Tyr-523, in expression vector pC0, were transfected into NIH-3T3 (contact-inhibited NIH Swiss mouse embryo) cells. The mutant induced cell transformation, recognizable by morphological changes as well as the capacity of the cells to form colonies on soft agar. The cells transfected with non-mutated c-fgr, which were selected by resistance to G418 (a neomycin derivative), were indistinguishable from untransfected or vector-transfected NIH-3T3 clones. Markers for differentiated myelomonocytic cells were tested for in transfected NIH-3T3 cells. Peroxidase, a representative enzyme of the mature granulocyte, was negative in all transfectants, but tests for 2-naphthyl butyrate esterase, a maker of mature monocytes, were strongly positive in clones transfected with normal c-fgr. The esterase reaction was completely inhibited by sodium fluoride, a specific inhibitor of the monocyte enzyme. The level of enzyme is dependent on the level of c-fgr expression. In contrast, cell clones transformed by mutant fgr showed only a minor population of esterase-positive cells. c-src, c-yes, fyn and lyn expression in NIH-3T3 cells was not correlated with esterase expression. The difference between normal and activated c-fgr genes with regard to esterase induction may be related to differencesin phosphorylation of target proteins. (K. Inoue, W. Bussabah, T. Akiyama & K. Toyoshima, unpublished work). A ckno wledgements Most of the work in this study was supported by a Grant in Aid for Special Project Research on Cancer Bioscience from the Ministry of Education, Science and Culture, Japan. We also thank Ms Yoko Sugiyama for preparation of the manuscript.
References Anderson, SK, Griffs CP, Tanaka A, Kung H-J, Fujita DJ 1985 Human cellular src gene: nucleotide sequence and derived amino acid sequence of the region coding for the carboxyl-terminal two-thirds of pp 60c-src.Mol Cell Biol 5 : 1122- 1129 Bjelfman C, Meyerson G, Cartwright CA, Mellstrom K, Hammerling U , Parlrnan S 1990 Early activation of endogenous pp60Src kinase activity during neuronal differentiation of cultured human neuroblastoma cells. Mol Cell Biol 10:361-370
src family protein tyrosine kinases
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Campbell M-A, Sefton BM 1990 Protein tyrosine phosphorylation is induced in murine B lymphocytes in response to stimulation with anti-immunoglobulin. EMBO F u r Mol Biol Organ) J 9:2125-2131 Cooper JA 1990 The src-family of protein-tyrosine kinases. In: Kemp B, Alewood P F (eds) Peptides and protein phosphorylation. CRC Press, Boca Raton, FL, p 85-1 13 Dymecki SM, Niederhuber JE, Desiderio SV 1990 Specific expression of a tyrosine kinase gene, blk, in B lymphoid cells. Science (Wash DC) 247:332-336 Glaichenhaus N, Shastri N, Littman DR, Turner JM 1991 Requirement for association of p56lCkwith CD4 in antigen-specific signal transduction in T cells. Cell 6451 1-520 Hirai H, Varmus HE 1990 Site-directed mutagenesis of the SH2- and SH3-coding domains of c-src produces varied phenotypes, including oncogenic activation of p6OC-"
Protein tyrosine kinases belonging to the src family Kumao Toyoshima*, Yuji Yamanashit, Kazushi Inoue*, Kentaro Sembat, Tadashi Yamamotof and Tetsu Akiyama* *Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565 and tlnstitute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108, Japan
Abstract. There are nine non-receptor-type protein tyrosine kinases that show a high level of similarity in their primary structures and in the structures of their functional domains. Together, they are called the src family. They seem to have common sites specific for oncogenic activation. Recent findings suggest that the kinases are closely associated with cell surface molecules and that they mediate extracellular signals through the activation of their tyrosine kinase activity. They appear to act more on the differentiated phenotype than in haemopoietic cell proliferation. Possible functions of the products of the Ick,fyn, lyn and fgr genes in lymphocytes and rnonocytes are discussed. I992 Interactions among cell signalling systems. Wiley, Chichester (Ciba Foundation Symposium 164) p 240-253
Protein tyrosine kinases (PTKs) are divided into two major categories-the receptor type and the non-receptor type. Kinases in the former category, represented by the product of the erbB/EGF receptor gene or the fms/CSF (colony-stimulatingfactor)-1 receptor gene, are encoded by several gene families. Similarly, the non-receptor tyrosine kinases are also encoded by at least four families; namely src, abl, fps/fes and src-kinase. The last enzyme is a novel ~ ~ product , of the PTK responsible for phosphorylation of Tyr-527 of P ~ O C - ~the c-src gene (Okada 8c Nakagawa 1989). PTKs in each family show structural similarity, as well as sharing genomic structure (such as that of the splicing junctions of the coding region; Toyoshima et a1 1987). However, the structure of the regions that regulate gene expression are specific to each gene. These similarities and specificities suggest that the members have common mechanisms for regulation of their enzymic activities, but have expression patterns that differ, possibly according to their particular function (Toyoshima et a1 1990). The biological significance of receptor-type PTKs has been widely discussed by many research groups, but the functions of the non-receptor type PTKs in normal states have been clarified only recently. The src gene, the representative of the src family, was known to be expressed in neuronal cells (Sudoh 1988), 240
src family protein tyrosine kinases
24 1
possibly playing a role in differentiation (Bjelfman et a1 1990), though the pathway of the signal is still unknown. The first evidence for a link between an external signal and a non-receptor type PTK was the association of p56Ick with CD4/CD8 antigen in T cells (Rudd et a1 1988, Veillete et a1 1988). Here, we describe significant aspects of the structure of the src family and discuss possible functions of some members of this family in haemopoietic systems. Structural similarity in the src family kinases Nine members of the non-receptor type PTK family are reported to have highly '~ 1). These kinases have conserved structure, typified by that of P ~ O C - ~ (Fig. molecular masses around 55 to 60 kDa and have a glycine residue at position 2, which is myristoylated after removal of the N-terminal methionine. This myristic acid and several amino acids next to Gly-2 are believed to be involved in the localization of the enzyme in the plasma membrane (Shulz et a1 1985). In fact, PTKs of this family are known to associate with membrane fractions; this is in contrast to the cytosolic or nuclear localization of other non-receptor type PTKs, such as Fps and Abl.
I
I GK
Y416
Y529
I
533
60.0
543
60.0
IG
I
I GK
Y
Y
I
529
59.5
IG
1
I GK
Y
Y
l
537
60.0
rG
I
I GK
Y
Y
1
512
50.6
IG
I
I GK
Y
Y
I
509
56.0
16
I
I GK
Y
Y
I
505
55.5
IG
I
I GK
Y
Y
]
499
55
?
?
FIG. 1. Schematic structures of six non-receptor-type protein tyrosine kinases. M, myristic acid; P, phosphate; G, glycine; K, lysine; Y, tyrosine. Numbers (right) indicate the number of amino acids and molecular mass in kDa Cfar right) of each kinase.
Toyoshima et al
242
EGFR p98f-fPs
SH2
1
p145c'ab1
P60c-src p45 gag-crk
PLC
GAP
527
UNIQUE SH3 gag
SH2
SH2
SH2 #H3
SH2 HSH
KINASE
R
b
SH2
a-SPECTRIN FIG. 2. A model of the functional domains of the src family tyrosine kinases. Additional proteins that have regions homologous to the modulatory domain are illustrated. EGFR, epidermal growth factor receptor; PLC, phospholipase C-y,; GAP, GTPase-activating protein.
Following the N-terminal sequence is a region of about 50 to 80 amino acids that is unique to each member-almost no homology is evident in this region. The difference in molecular size of the kinases is mostly due to the variation in the number of amino acids in this region. The region's function is still not clear, but it is postulated to play a role in association of the kinase with a surface molecule which is able to receive extracellular signals. The next region of the sequence, about 150 amino acids, has moderate similarity among members of the src family, and includes src-homologous (SH) regions. These SH regions were originally found as conserved regions in other non-receptor-type PTKs such as the abl and f p s gene products. This region is also conserved in a variety of cytoplasmic molecules that are thought to interact with PTKs (Fig. 2), and is considered to be the site that interacts with cytoplasmic proteins to modulate signals. The C-terminal half of the molecule is a highly conserved region; sequence identity is about 80 to 90%. This region includes the catalytic domain, which phosphorylates tyrosine residues of target proteins, and the C-terminal regulatory domain, which down-regulates the catalytic activity when the tyrosine at the most C-terminal position is phosphorylated. Similarity and dissimilarity of genomic structures The genomic organization of the coding regions of c-src is identical in the chicken and human (Takeya & Hanafusa 1983, Anderson et a1 1985). When the human c-fgr clone was analysed, the splicing junctions of exons 4 to 12 were
src family protein tyrosine kinases
243
found to coincide with those of the c-src gene. Similar coincidences of splicing junctions were also found in the c-yesgene (Nishizawa et al1985, 1986). Possible splicing junctions found in other members of the src family were also in the same locations as those of the c-src gene, except in the unique region. In contrast, splicing junctions in the c-erbB2 gene were completely different from those of the src family, even within the catalytic domain, suggesting that the nine members of the src family arose by duplication of a preformed ancestral gene. In contrast, regulatory elements located upstream of the src family genes are specific to each member of the family. This difference of regulatory elements may explain the unique pattern of each expression of src family genes in different tissues and organs. Oncogenic activation of the sre family genes The c-src gene, integrated into a retroviral genome, acquires cell-transforming capacity only after mutation. v-src, v-yes and v-fgr were found as oncogenes of acutely oncogenic retroviruses. All of these, including S1 and S2, new isolates containing activated c-src (Ikawa 1986), have lost a sequence encoding the Cterminal portion of their regulatory domains through recombination with viral or other cellular sequences. Thus, they have invariably lost a tyrosine residue at the most C-terminal position (corresponding to Tyr-527 of the c-src gene product). Substitution of phenylalanine for Tyr-527 also activated tyrosine kinase activity and added transforming capacity to p60c-src.In addition, substitution of an amino acid residue in the modulatory domain (Tyr-90 to Phe; Tyr-92 to Phe; Arg-95 to Glu, Lys or Trp) or in the kinase domain (Thr-338 to Ile; Glu-378 to Gly; Ile-441 to Phe), or deletions including part or almost all of the modulatory domain also confer transforming activity on p60c-src.These mutations are also known to activate the tyrosine kinase activity of the enzyme. Activation of other members of the family has not been tested extensively; deletion of C-terminal sequences including the tyrosine residue corresponding to Tyr-527 of p60c-src,or substitution of the same tyrosine by phenylalanine, activates both tyrosine kinase and transforming activities in c-Yes, c-Fgr, Fyn, Lck and Hck. Deletions in the modulatory domain and substitution of an amino acid in the kinase domain in Fyn are also reported to activate transforming capacity (Semba et a1 1990). These mutations and substitutions are summarized by Cooper (1990) and Semba & Toyoshima (1990). A precise analysis of mutations within the modulatory domain of p6WCwas published recently by Hirai & Varmus (1990). lck and fyn expression in T cells
ick was identified as a gene encoding a novel tyrosine kinase, p56Ick,that is abundantly expressed in mouse LSTRA (T cell lymphoma) cells (Marth et a1 1985, Voronova & Sefton 1986). Its human counterpart was soon found to be
244
Toyoshima et al
expressed in T lymphocytes. The dramatic decrease of p56/ckin T cells during their activation suggests that it plays an important role in this process (Marth et a1 1987). The protein appears to be functionally and physically associated with CD4 or CD8 in normal T cells, and cross-linking of CD4 activates the protein tyrosine kinase activity of p56lck(Veillette et a1 1988). Activation of T cells occurs when the T cell receptor is stimulated by a specific antigen and the CD4 antigen interacts with a class I1 major histocompatibility complex (MHC) molecule. The association of CD4 with p56ICk is critical in this process (Glaichenhaus et a1 1991). High expression of fyn was observed in T cells of mice with the autosoma1 recessive mutation Ipr (lymphoproliferation) or gld (generalized lymphoproliferative disease). These homozygous mice with autoimmune disease have polyclonal proliferation of CD4-/CD8- T cells and lymph node enlargement (Katagiri et a1 1989). Expression offyn is low in T cells of normal mice that have the same genetic background except for the lpr or gld gene. p56lckis expressed at similar levels in diseased and normal mice. The expression offyn in negatively selected CD4- K D 8 - T cells was increased 10-fold about two hours after stimulation by treatment with anti-CD3q concanavalin A, 12-O-tetradecanoylphorbol-13-acetate(TPA) or A23 187 (a calcium ionophore), while lck expression remained constant. In addition, p59fYn and CD3 were coprecipitated by anti-CD3. Thus, p56lckassociated with CD4/CD8 aids in the recognition of MHC molecules and p59fYn associated with CD3 is involved in the response to growth-stimulating treatment. CD45, a common lymphocyte antigen that has phosphotryosine phosphatase activity, is thought to activate p56ICktyrosine at position 505 (Ostergaard et a1 1989). Thus, at least three components may act in the regulation of T cell growth in response to a stimulus from outside the cell. Association of p56'Yn with the B cell antigen receptor complex Iyn is preferentially expressed in B cells in the lymphoid system (Yamanashi et a1 1989). p56'Yn is not readily precipitated as an immune complex by known antibodies to lymphocyte surface molecules. However, mild treatment with digitonin of WEHI-231 cells, a mouse B cell line, allowed us to co-immunoprecipitate p56bn with surface membrane-bound IgM. This loose association between p56IYn and the IgM can be expected because B cell antigen receptors have only a few amino acids in their cytoplasmic regions (Rogers et a1 1980). WEHI-231 cells produce two types of p chain-a membrane form (pM) and a soluble form (pS). pM was co-immunoprecipitatedwith p56/yfl by anti-Lyn, and IgM-associated molecules of 30 to 40 kDa were co-immunoprecipitated with p56bn by anti-IgM (Yamanashi et a1 1991). Cross-linkage of membrane IgM up-regulates the tyrosine kinase activity of ~56~Y",and phosphorylation at tyrosine residues of at least 10 molecules increases within
src family protein tyrosine kinases
245
0 0
om*. 0.0
Ick fY n
hCk
fgr
mast cell
lYn blk
src fgr l Yn
plasma cell macrophage stem cell
FIG. 3. cells.
Expression of cytoplasmic tyrosine kinases of the src family in haemopoietic
2-10 min after cross-linkage. As a result of this treatment p56'Yn was downregulated instantly, reaching a minimum level about 30 min after stimulation, whereas p53/Yn,which was also associated with IgM, was not down-regulated. These results, together with the recent finding that p56'vn and membrane IgD can be co-immunoprecipitated (Y. Yamanashi, T. Yamamoto & K. Toyoshima, unpublished work), suggest that p56bn plays an important role in antigendependent B cell differentiation. Although there is no evidence, CD45 and p5Sb" (Dymecki et a1 1990) may also play an important role in B cell proliferation and differentiation (Fig. 3). The connection between an 80 kDa tyrosine kinase described by Campbell & Sefton (1990) and p56[Yn is unclear at present.
246
Toyoshima et al
Possible functions of pSsfgr in monocytes
p58c-jgris known to be expressed in B cells infected with Epstein-Barr virus. Recently, we reported that the enzyme is specifically expressed in peripheral monocytes, granulocytes and natural killer cells. When HL-60, a promyelocytic cell line, was treated with retinoic acid or TPA to induce myelomonocytic differentiation, p5SC-fgraccumulated in differentiated cells (Inoue et a1 1990, Kawakami et a1 1988), suggesting that ~58~-fg' is more closely associated with differentiation than with proliferation. The c-fgr gene and a mutant encoding phenylalanine instead of Tyr-523, in expression vector pC0, were transfected into NIH-3T3 (contact-inhibited NIH Swiss mouse embryo) cells. The mutant induced cell transformation, recognizable by morphological changes as well as the capacity of the cells to form colonies on soft agar. The cells transfected with non-mutated c-fgr, which were selected by resistance to G418 (a neomycin derivative), were indistinguishable from untransfected or vector-transfected NIH-3T3 clones. Markers for differentiated myelomonocytic cells were tested for in transfected NIH-3T3 cells. Peroxidase, a representative enzyme of the mature granulocyte, was negative in all transfectants, but tests for 2-naphthyl butyrate esterase, a maker of mature monocytes, were strongly positive in clones transfected with normal c-fgr. The esterase reaction was completely inhibited by sodium fluoride, a specific inhibitor of the monocyte enzyme. The level of enzyme is dependent on the level of c-fgr expression. In contrast, cell clones transformed by mutant fgr showed only a minor population of esterase-positive cells. c-src, c-yes, fyn and lyn expression in NIH-3T3 cells was not correlated with esterase expression. The difference between normal and activated c-fgr genes with regard to esterase induction may be related to differencesin phosphorylation of target proteins. (K. Inoue, W. Bussabah, T. Akiyama & K. Toyoshima, unpublished work). A ckno wledgements Most of the work in this study was supported by a Grant in Aid for Special Project Research on Cancer Bioscience from the Ministry of Education, Science and Culture, Japan. We also thank Ms Yoko Sugiyama for preparation of the manuscript.
References Anderson, SK, Griffs CP, Tanaka A, Kung H-J, Fujita DJ 1985 Human cellular src gene: nucleotide sequence and derived amino acid sequence of the region coding for the carboxyl-terminal two-thirds of pp 60c-src.Mol Cell Biol 5 : 1122- 1129 Bjelfman C, Meyerson G, Cartwright CA, Mellstrom K, Hammerling U , Parlrnan S 1990 Early activation of endogenous pp60Src kinase activity during neuronal differentiation of cultured human neuroblastoma cells. Mol Cell Biol 10:361-370
src family protein tyrosine kinases
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Campbell M-A, Sefton BM 1990 Protein tyrosine phosphorylation is induced in murine B lymphocytes in response to stimulation with anti-immunoglobulin. EMBO F u r Mol Biol Organ) J 9:2125-2131 Cooper JA 1990 The src-family of protein-tyrosine kinases. In: Kemp B, Alewood P F (eds) Peptides and protein phosphorylation. CRC Press, Boca Raton, FL, p 85-1 13 Dymecki SM, Niederhuber JE, Desiderio SV 1990 Specific expression of a tyrosine kinase gene, blk, in B lymphoid cells. Science (Wash DC) 247:332-336 Glaichenhaus N, Shastri N, Littman DR, Turner JM 1991 Requirement for association of p56lCkwith CD4 in antigen-specific signal transduction in T cells. Cell 6451 1-520 Hirai H, Varmus HE 1990 Site-directed mutagenesis of the SH2- and SH3-coding domains of c-src produces varied phenotypes, including oncogenic activation of p6OC-"
