High glucose, insulin and palmitic acid triggers centrosome amplification.
Cell viability was not compromised by the treatment using high glucose, insulin and palmitic acid (Fig. 1A). Figure 1B was a representative image of CA. The experimental treatment increased the level of CA by approximately 6 times in the non-cancerous (NCM460) and cancerous (HCT116) colon cells (Fig. 1C).
The experimental treatment increases the binding between ROCK1 and KIF2A.
Upon the experimental treatment, the protein level of ROCK1 was increased at 12 and 24 hours, whereas, the protein level of KIF2A started to decrease at 24 hours (Fig. 2A). KIF2A was pulled down together with ROCK1 from the treated cells, but not from the control samples (Fig. 2B). ROCK1 was pulled down together with KIF2A in both control and treated sample. However, the level of KIF2A pulled down with ROCK1 from the treated cells was much higher than that from the control samples (Fig. 2C).
KIF2A is localized in the centrosome.
We reported that the experimental treatment increased the centrosomal translocation of ROCK1 (6). Here, we examined whether KIF2A was localized in centrosome. Indeed, as shown in Figure 3, KIF2A was present in only one centrosome at early stage of cell division, and then appeared in two centrosomes along the cell cycle progress. It was also clearly distributed in the spindles. When multipolar division occurred due to CA, KIF2A was present multipolar spindles.
Disruption of ROCK1-KIF2A complex attenuates the CA.
We used siRNA technology to knockdown the protein level either RCOK1 or KIF2A, attempting to inhibit the formation or disrupt the protein complex, and examined whether it inhibited the treatment-caused CA. Indeed, knockdown of ROCK1 (Fig. 4A) or KIF2A (Fig. 4B) did inhibit the CA (Figs 4C and 4D).
Molecular docking modeling for the interaction between ROCK1 and KIF2A.
Figure 5A showed the model of protein-protein interaction between ROCK1 and KIF2A, the score of which reached as high as 20.3. In ROCK1, 50 amino acids were likely to be involved in the interacting and maintaining the complex stability, which were VAL38, TYR39, ASP42, PHE43, PRO44., ALA45, LYS60, ASN64, LYS65, ARG67, ASP68, LYS72, ALA73, GLU74, TYR76, GLU77, VAL78, VAL79, LYS80, VAL81, ARG84, GLY85, ALA86, GLU89, GLU91, VAL 93, LYS96, MET104, LEU106, GLU111, LYS114, ASP145, ASP146, ARG147, TYR148, TYR150, SER166, ASP370, LEU371, GLU372, GLU373, ASP374, GLU377, GLU378, GLU379, THR380, PHR381, PRO382, ILE383, and LYS385. The key sequence pieces for the interacting were VAL38-ALA45, ASN64-LYS96 and ASP370-LYS385. There were 52 amino acids in KIF2A, which involved in the interacting, which were VAL188, ARG229, LYS230, ARG231, PRO232, LEU233, ASN234, LYS235, LYS236, GLU237, ASP281, ASP282, SER283, ALA284, PRO285, ASN286, GLU287, GLY316, SER317, GLY318, LYS319, THR320, HIS321, THR322, GLY234, GLY325, ASP326, SER335, LYS336, ARG344, LEU377, LEU378, ARG380, GLU408, ASP409, LEU411, LYS412, ASP415, ILE416, GLY417, ASN418, SER419, LYS420, ARG421, THR422, SER423, HIS431, SER432, SER433, ARG434, ASP457, and THR529. The key sequence pieces were ARG229-GLU237, GLY316-LYS336 and GLU408-ARG434. The surface structures of ROCK1 and KIF2A presented a highly complementary feature (Fig. 5B), which could form a dimer with strong stability. Moreover, we alignment of the sequences of ROCK1 and KIF2A revealed that the amino acids sequence similarity was only 16% (Fig. 5C).