MEP and A6fgr cells contained a number of tyrosine phosphorylated proteins detected by western blotting and immunoprécipitation. However, induction of the transfected c-fgr gene did not increase the number or amount of tyrosine phosphorylated proteins detected in A6fgr cells with the exception of a single protein, which is probably p55‘^^'^ itself.
The most likely explanation for the failure to detect increased tyrosine phosphorylation in A6fgr cells expressing p55®’^'' is that the protein was not active under these conditions. It is possible that hyper-expression of p55®"-^'' was not sufficient to overcome the normal regulatory mechanisms in myeloid cells which keep this enzyme inactive. A novel tyrosine kinase csk has been shown to down regulate the activity of other tyrosine kinases such as src, fyn, Jyn, Ick (Nada et al
1991, Hata et al 1994, Koegl et al 1994, Bergman et al 1992) and now c-fgr (Ruzzene et al 1994). The activity of p55‘"'^'’ was down regulated when Tyr523 was phosphorylated by csk and this then prevented autophosphorylation of Tyr412. Phosphorylation of both Tyr412 and Tyr523 occurred in the presence of polycationic peptides such as polylysine, histones and protoamines and resulted in the stimulation of catalytic activity (Ruzzene et al 1994). If the exogenous pSS®'-*'', present in A6fgr cells treated with cadmium ions, is phosphorylated primarily on Tyr523 then this may be responsible for keeping the protein in an inactive
conformation. It is interesting that in contrast with transfection into U937 cells, when c-fgr was transfected into the murine fibroblast cell line, NIH 3T3, the tyrosine kinase activity of p55®-^'' was apparent. It is possible that the normal regulatory mechanisms, such as csk or repressor protein (Ley et al 1989), were not active in these cells.
The tyrosine kinase activity of p55‘"'^'’ has been demonstrated in monocytes and neutrophils (Stefanova et al 1993, Hamada et al 1993, Morio et al 1994, Berton et al 1994), in HL60 cells treated with retinoic acid (Notario et al 1989, Miyazaki et al 1993), in a murine monocytic cell line (Hatakeyama et al 1994) and in NIH 3T3 cells transfected with various mutants of the c-fgr gene (Inoue et al 1991, Sartor et al 1991, 1992, 1993).
A 130KDa protein band was detected in c-fgr and c-fgr^^^ transfected clones and additional proteins of 65KDa, 80KDa and 70KDa were observed in clones transfected with c - f g i ^ ^ . Another band of 95KDa, in addition to the previous 130, 65, and 80KDa proteins, was detected in clones transfected with c-fgr^^^ and c-fgi^^^^. Other experiments identified a further band phosphotyrosine protein of 36KDa present in lysates of cells transfected with c-fgr^^^^, but not c-fgr (Sartor & Robbins 1993, see section 1.12). Sartor et al (1991) used an immune complex kinase assay to confirm that and p55^^^° were directly responsible for the phosphotyrosine proteins detected by western blotting. Incubation of anti- phosphotyrosine antibodies with lysates of NIH 3T3 cells transfected with c-fgr and c-fgr^^^^ resulted in immunoprecipitates which contained a tyrosine kinase capable of phosphorylating proteins of 130, 95, 85 and 60KDa.
Sartor & Robbins (1993) were able to identify five of these tyrosine phosphorylated proteins. They were GAP, pl25'^^, a 130KDa protein recognised by monoclonal antibody AF4, a 120KDa protein recognised by monoclonal antibody 2B12 and a 85Kda protein recognised by monoclonal antibody 4F11. A physical association
between and the 130KDa protein was demonstrated when anti-Fgr antiserum co-precipitated the 130KDa protein from cell lysates of NIH 3T3 transfectants containing (Sartor et al 1991, 1993). This 130KDa protein was then recognised by the AF4 monoclonal antibody (Sartor & Robbins 1993).
Miyazaki et al (1993) also examined tyrosine phosphorylation of cellular proteins in cells containing high levels of p55^:^\ Untreated HL60 cells contained a major tyrosine phosphorylated band of about 116KDa and several minor bands. During 96 hours of DMSO treatment, the 116KDa band disappeared and a doublet of about 170KDa appeared as a strongly tyrosine phosphorylated band. At the same time c- fgr expression and kinase activity increases, with maximal levels of protein and
p55‘"-^*'' kinase activity present after 48-72 hours of treatment.
In neutrophils, high levels of p55‘=-^*'' have been detected in cell fractions enriched for plasma membranes, secondary and tertiary granules. Activation of neutrophils by cytochalasin B and formyl-Met-Leu-Phe [fMLP] resulted in release of secondary and tertiary secretory granules, with subsequent loss of and kinase activity from these fractions. However, treatment with cytochalasin B alone, which resulted in only secondary granule release, did not change the level or activity of pSS'"-^'' in these fractions. Thus, p55^-^^'' was associated with the release of tertiary secretory granules by neutrophils in response to activation by fMLP (Gutkind & Robbins 1989).
In peripheral blood monocytes, p55‘"-^*'' kinase activity could be increased by stimulating cells with LPS (Stefanova et al 1993) or superantigen (Morio et al 1994). LPS is known to interact with CD 14, a GPI-linked protein found on the cell of monocytes (Golenbock et al 1993, Haziot et al 1993, Ziegler-Heitbrock & Ulevitch 1993). However, was not detected in anti-CD 14 immunoprecipitates suggesting that there is not direct physical association between p55':^^ and CD 14. Treatment of monocytes with toxic shock syndrome toxin-1
induced rapid activation of both p55‘^-^'' and p59^*as shown by autophosphorylation and phosphorylation of an exogenous substrate in an immune complex assay. A similar result was obtained with staphylococcal enterotoxin A and with crosslinking anti-MHC Class n antibodies (see section 4.3.3). Analysis showed that upon activation pSS®*-^'' was phosphorylated on 2 tyrosine residues, Tyr412 and a tyrosine residue in the N-terminal portion of the molecule.
Another protein that has been suggested as a target for pSS®'^'' is FcyRII. Hamada et al (1993) isolated human neutrophils from peripheral blood and showed that p55®-*'' was present in FcyRII immunoprecipitates. This association is discussed in more detail in chapter 6.
5.3.5. Conclusions.
The work presented in this chapter shows that hyper-expression of the transfected c-fgr gene in A6fgr cells did not effect the growth of the cells, their response to PMA, DHCC, TNFa or retinoic acid, their ability to adhere to fibronectin or the level of tyrosine phosphorylated proteins in the cell, other than p55®'^'’ itself. This is probably because the exogenous p55®-*'' was not active in A6fgr cells under these conditions. However, there were differences in the expression of some myeloid and adhesion antigens, notably an increase in ICAM-2 expression, a decrease in ICAM- 3 expression and an increase in ICAM-1 expression in the presence of DHCC. This suggests that c-fgr may play a role in controlling ICAM expression as well as signalling via ^ 2 integrins.
The tyrosine kinase activity of p55‘^-^'' has been demonstrated under a number of conditions : when transfected into fibroblasts, during myeloid differentiation, during ^ 2 mediated adhesion to fibrinogen and tertiary granule release in
neutrophils and in response to LPS and superantigen stimulation in monocytes. A number of different proteins are phosphorylated when p55®‘-*'’ is activated and some of these have been identified.
CHAPTER 6. THE ROLE OF C-FGR IN PHAGOCYTOSIS.