Differential regulation of T cell antigen responsiveness by isoforms of the src-related tyrosine protein kinase p59fyn.

Abstract

SllnunaryRecent observations suggest that the src-related tyrosine protein kinase p59~ may be involved in antigen-induced T lymphocyte activation. As a result of alternative splicing, p59~ exists as two isoforms that differ exclusively within a short sequence spanning the end of the Src Homology 2 (SH2) region and the beginning of the tyrosine protein kinase domain. While one p59~ isoform (fynB) is highly expressed in brain, the alternative product (fynT) is principally found in T lymphocytes. To further understand the role of p59~ in T cell activation and to test the hypothesis that p59~ T serves a tissue-specific function in T lymphocytes, we have examined the effects of expression of activated versions (tyrosine 528 to phenylalanine 528 mutants) of either form of p59~ on the physiology of an antigen-spedfic mouse T cell hybridoma. Our results demonstrated that the two forms offyn, expressed in equivalent amounts, efficiently enhanced antibody-induced T cell receptor (TCR)-mediated signals. In contrast, only p59~ T increased interleukin 2 production in response to antigen stimulation. This finding implies that the distinct p59~ isoform expressed in T lymphocytes regulates the coupling of TCtL stimulation by antigen/major histocompatibility complex to lymphokine production.ntigen-induced T lymphocyte activation is primarily mediated by changes in intracellular tyrosine protein phosphorylation (for review, see reference 1). Although the cellular machinery regulating this biochemical signal remains poorly characterized, recent observations suggest that the srerelated nonreceptor tyrosine protein kinases p56 ~ and p59~ are involved in this process, p56 lck is a lymphocyte-specific tyrosine protein kinase associated with and regulated by the CD4 and CD8 T cell surface antigens (for review, see reference 2). Increasing evidence demonstrates that a large part of the accessory function provided by CD4 or CD8 during T cell activation is mediated through p561r Contrary to p56 lCk, p59~ is expressed in most cell types (3-6). It is interesting that as a result of alternative splicing of mutually exclusive exons 7, p59~ exists as two isoforms differing solely within a stretch of 51 amino acids spanning the end of the SH21 region and the beginning of an otherwise classical tyrosine protein kinase domain (see Fig. 1 A) (7). It has been postulated that as a consequence of different substrate specificities and/or catalytic efl~ciencies, these two enzymes regulate distinct cellular processes.Whereas onefyn isoform 0CynB) accumulates highly in brain, the other (fynT) is expressed predominantly in T lymphocytes (7; L. M. L. Chow, A. Veillette, D. Davidson, unpublished data). The limited homology (53% or less) between the exon 7-encoded sequences offynT and those off, B, as well as of all other src-related tyrosine protein kinases, fostered the early view that p59~ T plays a specialized role in T lymphocyte physiology. The subsequent demonstration that a fraction of p59~ T is physically associated with elements of the TCR complex ...

The T cell enigma in lupus

“…Subsequently, it was found that the src nonreceptor protein tyrosine kinase (PTK), p59fy", may be mediating this effect, because it is overexpressed in DN T cells (36) (Figure 1). The significance of this novel observation is that the fyn kinase is associated with the CD3/T cell receptor (TCR) complex, and directly mediates the signal initiated by ligand binding (37,38) (Figure 1). It is interesting to speculate whether the constitutive tyrosyl phosphorylation actually reflects autophosphorylation of the fyn kinase.…”

PAG-Associated FynT Regulates Calcium Signaling and Promotes Anergy in T Lymphocytes

“…To extract PAG and PAG-associated proteins from lipid rafts, cells were lysed in a buffer containing maltoside (1% n-dodecyl-␤-D-maltoside, 50 mM Tris [pH 7.6], 150 mM NaCl, and 2 mM EDTA) supplemented with protease and phosphatase inhibitors (6). Immunoprecipitations and immunoblot analyses were performed as previously described (7,33). Radioactivity was quantitated with a Storm PhosphorImager (Molecular Dynamics, General Electrics Canada, Mississauga, Ontario, Canada).…”

Netrin 1 and Dcc regulate oligodendrocyte process branching and membrane extension via Fyn and RhoA

“…Primary antibodies used in this study were: rabbit polyclonal anti-netrin 1 PN3 (Manitt et al, 2001), mouse monoclonal anti-Dcc (G97-449; BD Biosciences Pharmingen, San Jose, CA, USA), goat polyclonal anti-Dcc (Santa Cruz Biotech, Santa Cruz, CA, USA), rabbit polyclonal anti-myelin basic protein (Mbp, Chemicon, Temecula, CA, USA), mouse monoclonal anti-Mbp (Chemicon), mouse monoclonal RIP antibody (Chemicon), mouse monoclonal anti-2Ј,3Ј-cyclic nucleotide 3Ј phosphodiesterase (Cnp, Sternberger Monoclonals, Lutherville, MD, USA), rabbit polyclonal antiMag (Chemicon), rabbit polyclonal anti-Nfm (Nefm -Mouse Genome Informatics) (Chemicon), rabbit polyclonal anti-Fyn (Upstate Cell Signaling, Charlottesville, VA, USA) used for western blots, rabbit polyclonal anti-Fyn [gift of Dr Andre Veillete and described previously (Davidson et al, 1992)] used for immunoprecipitation and western blots, mouse monoclonal anti-FAK (BD Biosciences), rabbit polyclonal anti-N-WASP (Santa Cruz), and rabbit polyclonal anti-phospho-Src (Cell Signaling), which recognizes the pY416 epitope in all SFK members.…”