Mutation of SPINOPHILIN (PPP1R9B) found in human tumors promotes the tumorigenic and stemness properties of cells

Abstract

Rationale: SPINOPHILIN (SPN, PPP1R9B) is an important tumor suppressor involved in the progression and malignancy of different tumors depending on its association with protein phosphatase 1 (PP1) and the ability of the PP1-SPN holoenzyme to dephosphorylate retinoblastoma (pRB). Methods: We performed a mutational analysis of SPN in human tumors, focusing on the region of interaction with PP1 and pRB. We explored the effect of the SPN-A566V mutation in an immortalized non-tumorigenic cell line of epithelial breast tissue, MCF10A, and in two different p53-mutated breast cancer cells lines, T47D and MDA-MB-468. Results: We characterized an oncogenic mutation of SPN found in human tumor samples, SPN-A566V, that affects both the SPN-PP1 interaction and its phosphatase activity. The SPN-A566V mutation does not affect the interaction of the PP1-SPN holoenzyme with pocket proteins pRB, p107 and p130, but it affects its ability to dephosphorylate them during G0/G1 and G1, indicating that the PP1-SPN holoenzyme regulates cell cycle progression. SPN-A566V also promoted stemness, establishing a connection between the cell cycle and stem cell biology via pocket proteins and PP1-SPN regulation. However, only cells with both SPN-A566V and mutant p53 have increased tumorigenic and stemness properties. Conclusions: SPN-A566V, or other equivalent mutations, could be late events that promote tumor progression by increasing the CSC pool and, eventually, the malignant behavior of the tumor.

Role of the Holoenzyme PP1-SPN in the Dephosphorylation of the RB Family of Tumor Suppressors During Cell Cycle

“…On the other hand, p107 and p130 also present the PP1 binding motif. The interaction between these two proteins and PP1 has been described in double hybrid assays and by co-immunoprecipitation [ 70 , 80 ]. However, this interaction seems to be much weaker and lasts less time than that between the pRB and PP1 [ 21 , 50 ].…”

“…SPN has been shown to be located in the cytoplasm and in the plasma membrane of cells but could also be expressed in the nucleus [ 91 , 100 ]. We recently demonstrated that SPN co-localizes with PP1α and PP1γ in the nucleus and in the cytoplasm of cells [ 80 ].…”

“…One of the main functions of SPN is to help PP1 dephosphorylate the pRB [ 28 , 80 , 93 ]. SPN interacts with PP1α and PP1γ but not with PP1β [ 69 , 80 ].…”

“…One of the main functions of SPN is to help PP1 dephosphorylate the pRB [ 28 , 80 , 93 ]. SPN interacts with PP1α and PP1γ but not with PP1β [ 69 , 80 ]. In addition, SPN interacts specifically with both total and phosphorylated pRB (P-pRB) in Ser807/811, two of the preferred PP1 dephosphorylation sites [ 10 , 80 ].…”

“…SPN interacts with PP1α and PP1γ but not with PP1β [ 69 , 80 ]. In addition, SPN interacts specifically with both total and phosphorylated pRB (P-pRB) in Ser807/811, two of the preferred PP1 dephosphorylation sites [ 10 , 80 ]. SPN is also able to bind and dephosphorylate phosphorylated p107 (P-p107) in Ser975, a homologous residue to Ser807/811 in P-pRB, and phosphorylated p130 (P-p130) in Ser672, an important residue implicated in the stability of p130 during the cell cycle and a possible dephosphorylation site of PP1 [ 20 , 34 , 53 , 54 , 80 ].…”