The Src homology 2 domain of the protein-tyrosine kinase p56lck mediates both intermolecular and intramolecular interactions.
A key event in signaling by many cell surface receptors is the activation of Src-like protein-tyrosine kinases and the assembly of protein complexes at the plasma membrane mediated by Src homology 2 and 3 (SH2 and SH3) domains. p56lck is a Src-related protein-tyrosine kinase which has SH2 and SH3 domains and is involved in T-cell signaling and oncogenic transformation. Here we demonstrate that purified recombinant SH2 and HSH3/SH2 domains of p56lck can mediate intermolecular interactions with a number of tyrosine-phosphorylated proteins present in lysates of NIH 3T3 cells transformed by a constitutively activated form of p56lck (p56lckF505). Two of the interacting tyrosine-phosphorylated proteins were identified as the p85 subunit of phosphatidylinositol 3-kinase and the GTPase-activating protein of p21ras. Using a synthetic phosphopeptide corresponding to the tyrosine-phosphorylated carboxyl terminus of p56lck (amino acids 494-509), purified recombinant Lck SH2 domain, and differentially phosphorylated forms of p56lck we provide evidence that the SH2 domain of p56lck can also mediate intramolecular interactions with the phosphorylated carboxyl terminus. Together these results suggest that the SH2 domain of p56lck has a dual function: (i) it can mediate intermolecular interactions with cellular proteins phosphorylated on tyrosine and thus might be involved in building up signaling complexes at the plasma membrane and (ii) it can bind to the tyrosine-phosphorylated carboxyl terminus of p56lck in an intramolecular fashion and thereby might be involved in the regulation of its intrinsic protein-tyrosine kinase activity. Phosphorylation/dephosphorylation of the regulatory tyrosine residue 505 might serve as a switch between these two functions.
Amrein
K E
KE
Department of Biology, Pharmaceutical Research-New Technologies, F. Hoffmann-La Roche Ltd., Basel, Switzerland.
Panholzer
B
B
Flint
N A
NA
Bannwarth
W
W
Burn
P
P
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002846:Chromatography, Affinity; D017931:DNA Primers; D020690:GTPase-Activating Proteins; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D051379:Mice; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D019000:Phosphotyrosine; D016133:Polymerase Chain Reaction; D011505:Protein-Tyrosine Kinases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D011506:Proteins / Q000032:analysis / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D014443:Tyrosine / Q000031:analogs & derivatives / Q000032:analysis
D017931:DNA Primers; D020690:GTPase-Activating Proteins; D010750:Phosphoproteins; D011506:Proteins; D011994:Recombinant Proteins; D019000:Phosphotyrosine; D014443:Tyrosine; D005982:Glutathione Transferase; D011505:Protein-Tyrosine Kinases; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck)