This study demonstrates an important role for the non-receptor tyrosine kinase Pyk2 in the integrin-mediated activation of PMNs that contributes to normal degranulation responses required for efficient host defense to S. aureus
infection. Pyk2 deficient PMNs exhibited reduced degranulation responses following integrin ligation both in vitro
and during bacterial infection in vivo
; however, they responded normally to soluble agonists suggesting that the integrin signaling pathway was the major response affected in the pyk2
mutant cells. It is clear that unlike Src-family or Syk tyrosine kinases, Pyk2 is acting in a more distal step in the integrin signaling pathway since many integrin mediated functions were normal in pyk2−/−
PMNs, including attachment, adhesion and integrin mediated activation of superoxide production. These limited impairments correlate with the only partially reduced integrin mediated tyrosine phosphorylation responses, though reduction in phosphorylation of specific substrates such as paxillin and Vav were observed. Our model for the role of Pyk2 in integrin-mediated activation shows it functioning downstream of both Src and Syk kinases to facilitate degranulation and migration responses through pathways involving paxillin and Vav (Supplemental Fig. 5
It is interesting that pyk2−/− PMNs showed no defects in in vitro responses to bacteria but were compromised in their ability to clear a bacterial infection in vivo. These differences can perhaps be attributed to the artificial nature of in vitro assays in general. In the in vitro bactericidal assays, there is a very large stimulus in the form of complement-opsonized S. aureus, which maximally activates complement receptors, TLRs and GPCRs at levels that are not likely to be present in vivo. Thus, in the in vitro system, only very gross defects are observed. However, more subtle defects, such as those observed with pyk2−/− mice, are revealed in the physiological in vivo system. It was in the in vivo model of infection that the defect in Pyk2-dependent integrin signaling, which resulted in impaired degranulation, impacted the bacterial clearance.
This report, which is the first examination of PMN function in pyk2−/−
mice, allows one to compare the effects of genetic deficiency of Pyk2 with chemical or peptide inhibitory approaches to study this enzyme (28
). Fuortes et al
demonstrated that the tyrphostin A9 inhibitor was the most potent blocker of integrin-mediated activation of PMN superoxide release among a panel of tyrosine kinase inhibitors and that treatment of cells with tyrphostin A9 resulted in loss of Pyk2 phosphorylation. This inhibitor also blocked PMN spreading, leading the investigators to conclude that Pyk2 participates in the signaling cascade leading to respiratory burst in TNF-treated adherent PMNs. However, it is unlikely that tyrphostin A9 is specific for Pyk2 since it was developed to inhibit PDGF receptor tyrosine kinase (41
) and also affects Ca2+
entry in CD3 stimulated Jurkat cells (42
). Using protein transduction of the COOH terminus of Pyk2 fused to a Tat peptide as a dominant negative inhibitor of Pyk2, Han et al
suggested that Pyk2 is required for TNF-mediated superoxide release as well as PMN spreading, confirming prior results with tyrphostin A9 inhibition. In contrast, the COOH-Pyk2/Tat fusion did not affect PMN degranulation in adherent PMNs or alter bacterial killing. These results, done with human PMNs, obviously differ significantly from our observations with Pyk2-deficient murine PMNs. There are many potential explanations for these disparate observations, such as potential compensation for Pyk2 deficiency by other signaling molecules, differences in experimental approaches or differences between human and murine cells. Though difficult to prove, it remains a formal possibility that the pyk2−/−
mutation generated in these mice may not be specific to pyk2
alone; ie the ES cells or mice themselves may have acquired other mutations.
Given that these studies were done with mice backcrossed for 8 generations, this reduces the probability that other mutations present in the ES cells may be contributing to this phenotype. Similarly, we found no differences in expression level of Src-family kinases, Syk, Vav, paxillin, Akt, Erk, p38, Rac, Rho, Cdc42, PAK1, or myosin light chain kinase (all signaling molecules in the integrin pathway) between WT and pyk2−/−
PMNs. Clearly there could be changes in other signaling molecules that could contribute to the PMN phenotype in pyk2−/−
mice; additional biochemical studies will be needed to sort out the exact pathways in PMN integrin signaling in which this kinase functions. Given that Pyk2-deficient PMNs showed a defect in degranulation responses, which very likely contributed to poor control of S. aureus
infection in vivo
, without a major defect in superoxide production, suggests that the pathways leading to these two responses are parallel and are not controlled by the same signals. This then leads to the question of how degranulation and exocytosis are controlled in PMNs following integrin-mediated activation. The complete molecular mechanism underlying control of degranulation by Pyk2 remains to be determined, however the dramatically impaired phosphorylation of paxillin and Vav observed in pyk2−/−
cells may provide clues. Both paxillin and Vav activation are associated with the activation and control of the Rho family GTPases. Paxillin can bind to the adaptor Crk which can lead directly to activation of the Rac GTPases following integrin-mediated adhesion (37
). Paxillin has also been shown to suppress the activation of RhoA. Similarly, Vav tyrosine phosphorylation is directly associated with activation of both the Rac and Rho GTPases (39
). Given the normal production of superoxide in pyk2−/−
PMNs following integrin ligation, it is unlikely that Rac2 is affected in these cells, since Rac2 is an important component of the NADPH oxidase in PMNs leading to superoxide production (43
). While several studies have suggested that Rac GTPases play roles in PMN degranulation in response to soluble agonists (44
), the potential role of Rac and Rho in regulating degranulation downstream of integrin ligation remains unclear. Using PAK-GST pulldown approaches and mAbs designed to recognize GTP-bound activated Rac, we were unable to demonstrate a significant impairment in Rac1 or 2 activation in pyk2−/−
deficient cells (data not shown). Examination of activation of downstream targets of Rac and Rho (PAK1 and MLC2, respectively) also showed no differences (data not shown). Hence it is possible that activation of other small GTPases via paxillin and Vav may be contributing to the degranulation phenotype we observed in pyk2−/−
cells. Paxillin also serves as an important scaffolding protein and has a direct affect on microtubule assembly (46
). Perhaps through paxillin phosphorylation, Pyk2 may contribute to microtubule polarization and assembly during integrin mediated degranulation responses, which remains to be investigated.
In summary, Pyk2 is playing a physiologically significant role primarily in integrin induced responses in PMNs. However, given its more downstream role compared to other non-receptor tyrosine kinases, Pyk2 deficiency does not produce as profound a defect in signaling as Src-family or Syk kinase loss. Hence, therapeutic targeting of Pyk2 may be useful in producing a more mild disruption in integrin function than blockade of Src-family or Syk kinases.