The fact that PEAK3 interacts with PYK2 suggests that, like PEAK2
[4][8][4,8], PEAK3 may activate specific TKs to mediate phospho-tyrosine signaling. We tested this hypothesis by co-expressing PEAK3 and PYK2 in HEK293T cells. While PEAK3 alone did not clearly affect protein tyrosine phosphorylation, PEAK3 robustly activated ectopic PYK2, as measured on the phosphorylation level of regulatory tyrosine 402 and 881 (
Figure 6a,c)
[26]. Interestingly, PYK2 activation was accompanied by an increase in cellular protein tyrosine phosphorylation, including a 50 KD and 120 KDa band (
Figure 6a). Pull-down experiments next identified PEAK3 and PYK2 some of these tyrosine phosphorylated proteins (
Figure 6b). Similar results were obtained in THP1cells where tyrosine phosphorylation of overexpressed PEAK3 was reduced upon acute pharmacological inhibition of PYK2 (PYK2i) (
Figure 6c,d). Additionally, the pharmacological inhibition of protein tyrosine phosphatases (pervanadate) largely increased PEAK3 tyrosine phosphorylation (
Figure 6c), suggesting the existence of regulatory protein tyrosine phosphatases. Consequently, PEAK3 tyrosine phosphorylation by PYK2 increased its association with GRB2 and ASAP1 (
Figure 6b), while PYK2i reduced this effect (
Figure 6c,d). ASAP1 was previously identified as a PYK2 substrate
[27] and, consistent with our findings, overexpressed PEAK3 increased ASAP1 tyrosine phosphorylation in a PYK2-dependent manner (
Figure 6e). Finally, this molecular process required an intact SHED module, suggesting that PEAK3 dimerization is essential for PEAK3 phospho-tyrosine signaling. Overall, these findings support a tyrosine phosphorylation-dependent PEAK3 scaffolding activity, which implicates the TK PYK2.
Figure 6. PEAK3 activates PYK2 signaling. (a) PYK2 activation by PEAK3 in HEK293T cells. The levels of protein tyrosine phosphorylation, PEAK3, PYK2, and phosphorylated PYK2 (pPYK2, pTyr402, and pTyr881) were assessed in cells transfected with the indicated constructs for 40 h (left). Relative quantification of PYK2 activation by PEAK3 (mean ± SD; n = 3); ** p < 0.01 (Student’s t test) (right). (b) PEAK3 tyrosine phosphorylation and its association with GRB2 and ASAP1 by PYK2 in HEK293T cells. The level-indicated proteins and their tyrosine phosphorylation in the PEAK3 pull-down were assessed in cells transfected with indicated constructs for 40 h. Relative quantification of PEAK3 tyrosine phosphorylation and its association with GRB2 and ASAP1 (mean ± SD; n = 3); ** p < 0.01, *** p < 0.001 (Student’s t test) (right). (c) PEAK3 tyrosine phosphorylation by PYK2 in THP1 cells. The pull-down of PEAK3 and its associated proteins were assessed in THP1 cells overexpressing PEAK3 and treated or not with pervanadate (PV) (0.1 μM for 15 min) or PYK2i (1 μM for 2h) as indicated. (d) Relative quantification of PEAK3 tyrosine phosphorylation and its association with GRB2 and ASAP1 in THP1 cells (mean ± SD; n = 3); * p < 0.05, ** p < 0.01 (Student’s t test) (right). (e) Increased ASAP1 tyrosine phosphorylation by PEAK3 expression in a PYK2-dependent manner. Immunoprecipitated ASAP1 tyrosine phosphorylation and its association with ST-PEAK3 from indicated THP1 cells treated as described in Figure 6c (n = 2).
This PEAK3 activating mechanism on PYK2 was then confirmed in PEAK3 overexpressing U2OS cells (Figure 7a). Importantly, this molecular effect was further enhanced in serum-starved conditions (Figure 7a), also supporting a growth factor-independent mechanism of PEAK3 activation of PYK2. PEAK3 knockdown reduced PYK2 activity in THP1 cells (Figure 7b), supporting the existence of a similar endogenous PEAK3–PYK2 signaling in leukemic cells. Having demonstrated the existence of PEAK3–AKT and PEAK3–PYK2 signaling, we then asked whether PYK2 would mediate the growth-independent AKT activation by PEAK3. Acute pharmacological inhibition of PYK2 (PYK2i) in U2OS reduced the stimulating effect of PEAK3 on AKT 3-fold, while this inhibitor had no effect on AKT activity in control cells (Figure 7c). Overall, these findings demonstrate the fact that that PEAK3 activates PYK2 as well as the support role of PYK2 activity in PEAK3–AKT signaling.
Figure 7. PYK2 activity mediates PEAK3–AKT signaling. (a) PYK2 activation by PEAK3 overexpressing U2OS cells that were serum-starved or not for >20 h. Western blots of the levels of PEAK3, PYK2, and pPYK2 (n = 2). (b) Regulation of PYK2 activity by endogenous PEAK3 in THP1 cells. PYK2 activity (pTyr402 PYK2 level) was measured in cells transfected with the indicated siRNAs. Quantification of PYK2 activity (mean ± SD; n = 3); * p < 0.05 (Student’s t test) (bottom). (c) PYK2 inhibition reduces PEAK3 activation of AKT in the absence of growth factors. U2OS cells that express or do not express PEAK3 were serum starved overnight or not, in the presence of PYK2 inhibitor (PYK2i) (1 μM) or vehicle (DMSO) as indicated. Western blots of the level of PEAK3, AKT, and pAKT (left) and relative quantification of AKT activity (mean ± SD; n = 3); ** p < 0.01 (Student’s t test) (right).