Floating cells were collected, washed free from nocodazole and replated in fresh medium. After the indicated time, cells were harvested and processed for immunoblotting and FACS analysis. Activation of NDR1/2 was accessed using anti-T444- P, NDR1 and NDR2 antibodies. Cell cycle distribution was accessed using PI staining and FACS analysis. Cell cycle phases were confirmed using Cyclin B1, Cyclin D1 and Cyclin A expression. B: HeLa cells were arrested at G2/M-border using nocodazole treatment for 14h. Cells were released into M/G1 phase for the indicated time before harvesting. Lysates were subjected to immunoblotting and immuno-precipitation of endogenous NDR species using a mixture of isoform specific antibodies. NDR kinase activity was accessed using peptide kinase assay (n=3). C: HeLa cells were transfected with dominant-negative MST1, MST2 or MST3 and 24h later arrested with nocodazole for 14h. Arrested cells were harvested or released into G1 for 8h before harvesting. NDR activation was accessed using T-444-P antibody. Cell cycle phases were confirmed by analyzing Cyclin B1 and p27 expression. D: HeLa cells were transfected with control siRNA (siC) or siRNA against MST3 (siMST3) and treated and analyzed as described in C. MST3 activation was accessed by using a P-MST4- T178/-MST3-T190/-STK25-T174 specific antibody (anti-P-MST3). Note that the P-MST3 signal disappears in the siMST3 treated samples.
in G1-phase of the cell cycle with the activation persisting until the end of S-phase. The G1-activation of NDR1/2 was confirmed by analyzing endogenous NDR1/2 activity using a peptide kinase assay (Figure 1B). HeLa cells were arrested at G2/M-
border and either harvested directly or released for 8h into fresh medium. Endogenous NDR species were immuno-precipitated and their activity measured using a specific peptide kinase assay. Direct assessment of NDR kinase activity confirmed that NDR kinases were activated in G1, whereas the activity was decreased in M-phase as compared to unsynchronized cells (Figure 1B).
Three members of the mammalian STE20 like kinases have been implicated in the regulation of NDR1/2: MST1, MST2 and MST3 (11, 13, 14, 19). For each MST kinase it has been shown that the kinase-dead variant can act in a dominant negative manner (14). To test which of the MST kinases was important for NDR1/2 activation in G1, we analyzed G1 activation of NDR upon over-expression of DN-MST1-3 (Figure 1C). Strikingly, although over-expression of DN-MST1 and DN-MST2 resulted in a moderate decrease in NDR activation, the expression of DN-MST3 significantly reduced NDR phosphorylation in this setting. To validate this finding we knocked-down endogenous MST3 using siRNA-technology and analyzed NDR activation (Figure 1D, Figure S1A). Depletion of endogenous MST3 resulted in decreased NDR activation in G1, confirming the role of MST3 for G1-activation of NDR1/2. To test whether MST3 phosphorylation would be increased in G1, MST3 activity was analyzed in our setting (Figure 1D, Figure S1A). Importantly, when comparing P-MST3 levels in G1 phase cells versus M-phase arrested cells, an increase of P-MST3 was observed in G1, which was not observed in cells depleted from MST3. Taken together, NDR kinases were activated in G1-phase of the cell cycle, with the activation persisting until late S-phase. Furthermore, our experiments revealed MST3 as the responsible upstream kinase for NDR1/2 in this setting, providing the first functional link between NDR and MST3.
Decrease in NDR1/2 results in cell proliferation defects due to a G1 block
To analyze whether NDR kinases functioned in cell cycle progression and proliferation we made use of HeLa cells expressing shRNA against NDR1 and NDR2 (Figure 2A). Both isoforms were targeted to avoid any compensatory effects as described earlier (Cornils, et al.; submitted). Knock-down of NDR kinases resulted in consistent proliferation defects of around 50%, which were not observed in control clones expressing shRNA against firefly luciferase (Figure 2B/C). In addition the effects of single knock-down of either NDR1 or NDR2 were tested (Figure S2). Interestingly, no compensatory effects of knocking down one isoform were observed (Figure S2C). Although compensation of NDR1 deficiency by NDR2 in healthy tissues was described earlier (Cornils, et al.; submitted), this data suggested that in our setting the compensatory mechanisms regulating NDR kinase isoforms were disturbed. Next we tested whether the observed proliferation defects would be a result of a cell-cycle block. Using BrdU incorporation we observed an increase in cells in G1 accompanied by a decrease in S-phase in NDR1/2 depleted cells (Figure S3). As normal proliferating HeLa cells were mainly in G1, we employed a method described by Mikule et al. to verify G1 blockade (18). Before cell-cycle analysis, nocodazole was added for 14h to accumulate cycling cells at the G2/M-border (Figure 2D). Indeed, cells deprived of NDR mostly stayed in G1, confirming that knock-down of endogenous NDR kinase species resulted in G1-arrest. An earlier report showed that NDR kinases were important for centrosome duplication (12). Disturbances in centrosome assembly have been reported to trigger the activation of a centrosome integrity checkpoint resulting in the activation of p38 and p53 with subsequent upregulation of p21 and G1-arrest (18, 20). Furthermore, inhibiting p38 in this setting decreased G1-arrest. To test whether the centrosome integrity checkpoint was
activated by depleting NDR1/2, we treated the cells with inhibitors for p38 prior to G1-arrest assessment. Adding p38 inhibitors to cells deprived of NDR did not result
Figure 2. shRNA mediated knock-down of NDR1/2 results in defects in cellular proliferation due to a G1-block.