Pyk2 and Syk participate in functional activation of granulocytic HL-60 cells in a different manner

The roles of the protein tyrosine kinases Pyk2 (also called RAFTK or CAK be ta) and Syk in the process of functional activation of human myeloid cells were examined. During granulocytic differentiation of HL-60 cells with dime thyl sulfoxide (DMSO), the amounts of Pyk2 and beta 2 integrin increased, w hereas the amount of Syk was abundant before differentiation and did not ch ange during differentiation. When the granulocytic cells were stimulated wi th N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), tyrosine phosphory lation of Pyk2 occurred promptly and subsequent association of Pyk2 with be ta 2 integrin was detected. In contrast, Syk was not tyrosine phosphorylate d by fMLP Introduction stimulation but constitutively associated with beta 2 integrin, Stimulation with fMLP also caused the alteration of beta 2 inte grin to an activated form, a finding that was confirmed by the observation of fMLP-induced cell attachment on fibrinogen-coated dishes and inhibition of this attachment by pretreatment with anti-beta 2 integrin antibody. Cell attachment to fibrinogen caused the enhanced tyrosine phosphorylation of P yk2 and the initial tyrosine phosphorylation of Syk, which was also inhibit ed by pretreatment with anti-beta 2 integrin antibody. In vitro kinase assa ys revealed that Pyk2 and Syk represented kinase activities to induce tyros ine phosphorylation of several molecules in the anti-beta 2 integrin immuno precipitates of the attached cells. These results showed that Pyk2 is invol ved in the functional activation of granulocytic cells in 2 signaling pathw ays: an fMLP receptor-mediated "inside-out" signaling pathway that might ca use beta 2 integrin activation and a subsequent beta 2 integrin-mediated "o utside-in" signaling pathway. Syk was activated in relation to cell attachm ent to fibrinogen as a result of "outside-in" signaling, although it was al ready associated with beta 2 integrin before fMLP stimulation. (C) 2000 by The American Society of Hematology.