SHP-1 plays an important role in negative signaling in many cell types. For example, after BCR stimulation in apoptotic B cells, SHP-1 has been shown to be recruited to phosphorylated ITIMs present in receptors such as CD72. However, the SHP-1 substrates in the chicken B cell line, DT40, have been poorly undefined. To identify SHP-1 substrates in DT40, we used a trapping mutant SHP-1 C/S (a catalytically inactive form). BCR stimulation induced hyper-phosphorylation of 230 kDa protein in C/S transfectants. MALDI-TOF/MS analysis revealed that this was myosin carrying ITIM. SHP-1 was shown to bind to this ITIM in synthetic peptide binding experiment. Thus, myosin is a direct SHP-1 substrate in B cells. The results suggest that SHP-1 plays a critical role in the reorganization of cytoskeletal architecture mediated via BCR stimulation. (C) 2003 Elsevier Science (USA). All rights reserved.