In this report we provide evidence that signalling to release of arachidonate induced in resident mouse peritoneal macrophages by non-opsonized zymosan (yeast cell-wall particles) and the Gram-positive bacterium S. aureus, is differentially dependent on SFK and the Tec kinase Btk. Src kinases act upstream of both Btk and the MAP kinases ERK and p38, thereby also of activating phosphorylation(s) of cPLA2. They are also most likely responsible for the tyrosine phosphorylation of PLCγ2 that occurs in response to zymosan, as shown here and previously . Btk is important for arachidonate release, but independent of the MAP kinase cascade.
The major enzyme responsible for release of arachidonate in the cells used in the present study is cPLA2, which is regulated by both phosphorylation(s) and an increase in intracellular Ca2+ [1, 2]. Several sites on cPLA2, especially in the C-terminal cluster of serine residues , become phosphorylated upon agonist stimulation and the protein kinase Mnk-1 has been suggested to be involved in the phosphorylation of one of these (Ser 727) . Our finding that a direct inhibitor of this kinase reduced zymosan-induced arachidonate release is consistent with the suggestion. Mnk-1 is coordinately regulated by the MAP kinases ERK and p38 and inhibition of both of these MAP kinases severely inhibits zymosan-induced arachidonate release . However, separate inhibition of either of the two kinases argues for a more prominent role for ERK than p38 . The SFK inhibitor PP2 counteracted bacteria- and zymosan-induced phosphorylation of both ERK and p38. PP2 has been shown to inhibit human p38 with similar potency as the SFK member Lck  which could, potentially, influence the interpretation of our data on arachidonate release. However, the pronounced inhibition by PP2 of the activation of MAP kinases, including p38, makes any direct inhibitory effect on p38 subordinate. A similar inhibitory effect on MAP kinase phosphorylation/activation was exerted by SKI-1, as illustrated by its effect on ERK. SFK are previously known to regulate MAP kinase activation (see  for review). In contrast, inhibition of Btk did not inhibit the MAP kinase cascade.
PI3K has an important role in zymosan- and bacteria-induced signalling in macrophages . SFK apparently affect the zymosan-induced phosphorylation of Akt, a downstream kinase of PI3K, indicating that one or more of these tyrosine kinases are situated upstream of PI3K. SFK and PI3K may interact in several ways; it is known that the p85 subunit of PI3K is able to interact with both the SH3 and SH2 domain of Src and it is known that the p85 subunit can function as a substrate for SFK . Furthermore, the p85 subunit of PI3K is known to interact with phosphotyrosine residues on different adaptor proteins. Binding of PI3K to such residues or a tyrosine kinase at the membrane is likely to help position the catalytic subunit of PI3K to its lipid substrate.
We now demonstrate that the tyrosine phosphorylation of PLCγ2 induced by zymosan is dependent on SFK, as shown by its sensitivity to the inhibitors PP2 and SKI-1. PLCγ is a possible substrate for Src  and the activation of PLCγ was blocked by PP1 (another Src kinase inhibitor) both in muscle cells from chicken embryos  and in FDC-P1 cells stimulated by EPO . Furthermore, Src activation has been shown to induce calcium release via a PLCγ dependent mechanism in Xenopus egg extracts . These results all indicate that SFK are important regulators of PLCγ. Because the phosphorylation of PLCγ2 is insensitive to wortmannin, as shown here as well as previously , the effect of PP2 is probably not mediated through PI3K but either direct or mediated by another kinase. It should be emphasized, though, that the role of tyrosine phosphorylation of PLCγ2 in the regulation of its activity remains unclear (see  for review). S.aureus did not induce detectable tyrosine phosphorylation of PLCγ2. Nevertheless both phosphorylation of ERK and arachidonate release induced by this bacterium were sensitive to wortmannin (data not shown) and therefore most likely mediated via PI3K and accompanied by activation of PLCγ2.
We also found that inhibition of Btk did not affect the zymosan-induced tyrosine phosphorylation of PLCγ2 in macrophages. Most studies on Btk have been carried out in B cells, while information about the role of Btk in macrophage signaling is scarce. Btk activation in B-cells is known to affect both PI3K and Ca2+ levels and Btk activation results in a rise in the level of IP3 and depletion of intracellular calcium stores . Furthermore, Btk regulates PtdIns4,5P2 synthesis which may affect both Ca2+-signaling and PI3K activity . Btk can associate with PtdIns4P-5kinases, enzymes that synthesize PtdIns4,5P2, and upon activation generate local PtdIns4,5P2 synthesis . PtdIns4,5P2 is a substrate not only for PI3K but also for PLCγ2 and increased synthesis may well be necessary to provide substrate for PLCγ2 and also be of importance for optimal generation of PtdIns3,4,5P3 . A difference between the phosphorylation of PLCγ2 by zymosan in macrophages and its phosphorylation after B cell receptor cross-linking is the PI3K dependency. Zymosan-induced PLCγ2 phosphorylation is not inhibited by wortmannin , whereas PLCγ2 phosphorylation in B cells is PI3K dependent [26, 28]. In view of our own data and the findings referred to above, we propose that Btk may primarily affect arachidonate release via the generation or further metabolism of PtdIns4,5P2 and the cellular Ca2+-homeostasis.