Conservation of the SEPT7 phosphorylation site among species
Our previous study shows that PKA phosphorylates SEPT12 at Ser196, which locates at the GTP-binding domain (GBD), thus disrupting septin filaments in the sperm annulus . GBD is conserved among septin family, we thus screened the PKA recognition motif, [R/K]-X-X-[pS/T], using amino acid alignments of 28 selected septin sequences among all septin subgroup. This PKA recognition motif was identified in most septins, except for SEPT1, SEPT5, SEPT6 and SEPT8 (Fig. 1A). Here we were interested in SEPT7 as it was the irreplaceable component of all septin filaments . The putative PKA phosphorylation site of human SEPT7 located on the Thr197, we thus checked whether this PKA phosphorylation site was conserved among different species. From zebrafish to homosapien, the predicted phosphorylation site is highly conserved (Fig. 1B). Thus, Thr197 of SEPT7 was a putative phosphorylation site that targeted by PKA.
SEPT7 Is Phosphorylated By PKA
Next, we checked whether PKA was responsible for SEPT7 phosphorylation at Thr197. Due to lack of antibody specifically against phosphorylated SEPT7 on Thr197, alternatively, FLAG-tagged SEPT7 was transfected into cells and the cell lysates were analyzed by immunoblotting with antibody against phospho-Threonine following immunoprecipitation with FLAG antibody. Treatment of 8-Br-cAMP, the activator of PKA, increased the phosphorylation on all Thr sites of SEPT7 in 293T (Fig. 2A) and NT2/D1 (Fig. 2B) in a dose dependent manner. This phosphorylation signal was reduced when cells were transfected with phospho-deficient SEPT7 (T197A) mutant. These results suggested that activation of PKA increased SEPT7 phosphorylation at Thr197 site. To further confirm this, the PKA catalytic subunit, PKACA2, was co-transfected with wild-type SEPT7 or T197A mutant and the phosphorylation status was observed. As expected, overexpression of PKACA2 increased SEPT7 phosphorylation at Thr and this signal was reduced in T197A mutant of 293T and NT2/D1 cell lines (Fig. 2C-D). Thus, PKA phosphorylates SEPT7 on Thr197.
SEPT7 Interacts With PKA Via The GTP-binding Domain
Then we checked whether SEPT7 interacted with PKACA2. FLAG-tagged SEPT7 and HA-tagged PKACA2 were co-transfected into 293T and NT2/D1 cell lines followed by performing immune-precipitation assay. PKACA2 was detected in the precipitant of FLAG-SEPT7 and vice versa (Fig. 3A), suggesting that SEPT7 interacted with PKACA2. Next, the interacting domain of SEPT7 was examined. The SEPT7 was dissected into N-terminus (N’), GTP-binding domain (GBD), and the C-terminus (C’). GFP-tagged GBD, N’, or C’ were co-transfected with HA-tagged PKACA2 into 293T or NT2/D1 cell lines followed by performing immune-precipitation assay. PKACA2 was detected in the precipitant of GFP-tagged GBD (Fig. 3B), but not in N’ or C’ precipitant (Fig. 3C-D), and vice versa. Thus, SEPT7 interacted with PKACA2 via the GBD.
SEPT7 Phosphorylation Disrupts Septin Filament Formation
SEPT7 is crucial for septin filament oligomerization, we thus checked whether SEPT7 phosphorylation affected septin filament formation. Two human SEPT7 mutant constructs, the T197E and T197A, which mimetic the constitutive phosphorylated and de-phosphorylated status, respectively, were generated and their effects on septin filaments was observed. The SEPT7 filaments were observed throughout the cytoplasm when cells were transfected with wild-type SEPT7 (Fig. 4A, upper panel, and 4B). This phenotype was also observed in cells transfected with the T197A (Fig. 4A, middle panel, and 4B). However, when cells were transfected with T197E, the septin filaments were hardly to be detected and smear signal was shown throughout the cytoplasm (Fig. 4A, lower panel; and 4B), suggesting that phospho-memetic SEPT7 did not form filaments. Next, cells were co-transfected with wild-type and T197E constructs and the septin filaments were examined. Wild-type SEPT7 showed filamentous structures in the cells. However, when cells were co-transfected with T197E, the population of cells with septin filaments was decreased in a dose dependent manner (Fig. 4C), suggesting T197E had dominant-negative effect in SEPT7 filament formation. Thus, phosphorylation of SEPT7 disrupts septin filament formation.
SEPT7 Phosphorylation Disrupts SEPT7/ SEPT7 Interaction
Septin filament was orchestrated by the order of the septin hexamer core complex arranged as SEPT2-6-7-7-6-2 or SEPT4-6-7-7-6-4  (Fig. 5A). SEPT7 phosphorylation disrupted septin filaments, thus, the interactions of SEPT7 with different septins were examined. Different septin constructs were transfected into NT2/D1 cells followed by performing immunoprecipitation assay. The interactions of FLAG-tagged SEPT7, including wild-type, T197A, or T197E, with Myc-tagged SEPT6, HA-tagged SEPT4, or HA-tagged SEPT2 were then examined by immunoblotting assay. First, we checked whether the interaction between SEPT7, which was important for polymerization of the septin complex, was affected by SEPT7 phosphorylation. FLAG-tagged SEPT7 and GFP-tagged SEPT7 were co-transfected into cells, the FLAG-SEPT7 was then precipitated by anti-FLAG antibody. GFP-SEPT7 could be detected in either wild-type or T197A precipitant (Fig. 5B). However, this interaction was hardly to be detected in the precipitant of T197E mutant (Fig. 5B), suggesting that SEPT7 phosphorylation disrupted SEPT7-SEPT7 interaction. Next, the SEPT2-6-7 and SEPT4-6-7 complex were checked. The SEPT2 and SEPT6 were detected in wild-type SEPT7 or T197A precipitant or T197E mutants, suggesting that SEPT7 phosphorylation did not affect the orchestration of SEPT2-6-7 hetero-complex (Fig. 5C). SEPT7 phosphorylation status also did not affect SEPT4-6-7 hetero-complex (Fig. 5D), suggesting that SEPT7 phosphorylation affected SEPT7-SEPT7 interaction. Our immunofluorescence data suggested that SEPT7 T197E was a dominant-negative mutant on septin filament polymerization. To further confirm the dominant-negative effect of T197E, cells were co-transfected with equal amount of wild-type SEPT7 (GFP-tagged SEPT7 or HA-tagged SEPT7) and different amounts of FLAG-tagged SEPT7 T197E, and the interaction between GFP-SEPT7 and HA-SEPT7 was examined. In the absence of T197E, GFP-SEPT7 interacted with HA-SEPT7 (Fig. 5E). However, with the increasing amount of T197E, the interaction between GFP-tagged SEPT7 and HA-tagged SEPT7 was reduced, supporting thta T197E was a dominant negative mutant on the assembly of septin filament. Thus, SEPT7 phosphorylation blocks SEPT7-SEPT7 interaction, but not affects SEPT2-6-7 or SEPT4-6-7 complex interaction.
Then the effect of SEPT7 phosphorylation on different septin filaments were examined by immuno-fluorescent staining. Wild-type SEPT7 and T197A mutant formed filaments, and these SEPT7 filaments were co-localized with SEPT2, SEPT4, SEPT6, and SEPT7, respectively (Fig. 6A-D, upper and middle panels). However, the T197E mutant showed dispersed signal in the cytoplasm, and no SEPT2, SEPT4, SEPT6, and SEPT7 filaments were observed (Fig. 6A-D, lower panel), suggesting that T197E mutant disrupted septin filaments. Taken together, SEPT7 phosphorylation blocks SEPT7-SEPT7 interaction thus disrupting septin filament formation.
Overexpression Of PKA Disrupts SEPT7 Filament Formation
Our data showed that SEPT7 was phosphorylated by PKA and SEPT7 phosphorylation disrupted septin filaments. Then we checked whether overexpression of PKA affects septin filaments. SEPT7 showed filamentous structures in 293T or NT2/D1 cells, however, these SEPT7 filaments became fainted and were disrupted when PKACA2 was overexpressed (Fig. 7A). Then, we checked whether overexpression of PKACA2 affected SEPT7-SEPT7 interaction. In the absence of PKACA2, SEPT7 interacted with SEPT7. However, this interaction was reduced when PKACA2 was overexpressed (Fig. 7B), supporting that PKACA2 blocked SEPT7-SEPT7 interaction. Then, the SEPT2- or SEPT4-SEPT6-SEPT7 complexes were examined. SEPT7 interacted with SEPT6, SEPT2 and SEPT4, and these complexes were not affected when PKACA2 was overexpressed (Fig. 7C-D). Thus, PKACA2 reduces SEPT7-SEPT7 interaction followed by disrupting septin filament formation.
PKA-mediated SEPT7 phosphorylation affected ciliogenesis
Septin-based ring filaments act as a diffusion barriers at the base of primary cilia . We then investigated whether SEPT7 phosphorylation modulated primary cilia formation. Under serum starvation, cells start to grow primary cilia , we thus checked whether serum starvation affected SEPT7 phosphorylation. Under serum starvation, the phosphorylation of SEPT7 was reduced (Fig. 8A), suggesting that SEPT7 phosphorylation might affect ciliogenesis. To further confirm our hypothesis, different SEPT7 mutants were transfected into RPE1 cells, the model cells for ciliogenesis, and the population of cells with primary cilium was quantified. Transfection of T197A had no effect on ciliogenesis when compared with transfection of wild-type SEPT7. However, the frequency of ciliated was reduced when RPE1 cells were transfected with T197E (Fig. 8B). Thus, SEPT7 phosphorylation inhibits primary cilia formation. Then, the role of cAMP-PKA cascade was examined. Treatment of cAMP reduced the frequency of ciliated and the length of cilia (Fig. 8C-E). In addition, overexpression of PKA inhibited ciliogenesis significantly (Fig. 8F). Thus, SEPT7 phosphorylation inhibits ciliogenesis during serum starvation.