Phosphorylation site mutated RB exerts contrasting effects on apoptotic response to different stimuli

Abstract

The retinoblastoma tumor-suppressor protein (RB) is an important regulator of cell cycle and apoptosis. RB is phosphorylated by cyclin-dependent protein kinase during cell cycle progression. A phosphorylation site mutated (PSM)-RB has previously been shown to cause G1 arrest and to interfere with S phase progression. In this study, we examined the effect of inducible PSM-RB expression on the apoptotic response to three different death stimuli: doxorubicin (DOXO), staurosporine (STS) and tumor necrosis factor (TNF) in Rat-16 cells. Induced expression of PSM-RB attenuated caspase activation by DOXO as a result of cell cycle arrest. STS has been shown to cause RB-dependent G1 arrest or apoptosis; however, expression of PSM-RB did not prevent caspase activation by STS. Surprisingly, induced expression of PSM-RB stimulated the apoptotic response to TNF in Rat-16 cells, which mostly undergo necrosis in the absence of PSM-RB. These results show that PSM-RB exerts disparate effects on apoptotic response to different stimuli, and that cell cycle arrest does not always associate with resistance to apoptosis.

Retinoblastoma family genes

“…Some of the ability of Rb to limit apoptosis may be due to its ability to control the cell cycle: in response to DNA-damaging agents quiescent cells are more resistant to apoptosis than dividing cells, and overexpression of Rb is similar in effect to other modifications that arrest the cell cycle (Chau and Wang, 2003;Masselli and Wang, 2006). Similarly, models have proposed that the induction of apoptosis in RbÀ/À cells is indirectly due to their continued proliferation as they initiate differentiation, and that this conflict signals for apoptosis.…”

Dephosphorylation of threonine-821 of the retinoblastoma tumor suppressor protein (Rb) is required for apoptosis induced by UV and Cdk inhibition

“…The data showing that phosphorylated Rb inhibits apoptosis is consistent with this idea. [10][11][12] Further, the observation that Rb is dephosphorylated in apoptosis induced by various stimuli also supports a required role for Rb dephosphorylation in apoptosis. [13][14][15][16][17][18][19] Because it was unclear which phosphorylation sites of Rb were involved in the apoptotic signal, we utilized glutamic acid mutagenesis to individually change each Rb phosphorylation site to glutamic acid, to mimic the phosphorylated state.…”

“…9 The mechanism of Rb regulation of apoptosis has not been fully elucidated; however, hyperphosphorylation of Rb has been shown to be important for its ability to inhibit apoptosis. [10][11][12] Loss of phosphorylation of the Rb protein has been observed during apoptosis. [13][14][15][16][17][18][19] Also, the specific activation of Rb-directed phosphatase activity has been shown to be required for apoptosis to occur.…”

“…In fact, Rb that lacks nine of the 16 phosphorylation sites, called PSM-RB (phosphorylation site-mutated Rb) does not protect cells from cell death initiated by apoptotic stimuli. 12 Thus, it seems likely that phosphorylation at specific sites on Rb is required for Rb to be able to block apoptosis. In the aforementioned study, however, there was no analysis of which specific Rb phosphorylation sites are involved incubation, cells were analyzed by immunoblotting.…”

Cytoskeletal Organization and Rb Tumor Suppressor Gene

“…Mutations of nine of these consensus phosphorylation sites, including seven sites at the C-terminal and two sites at the insert region of Rb, are sufficient to constitutively active Rb and block DNA replication (52)(53)(54). Also, mutations of this phosphorylation site can cause different cell cycle and apoptotic effects in Rat-16 cells exposed to various stimuli, such as tumor necrosis factor, doxorubicin or staurosporine (55). In addition, Rb may mediate DNA damage response (DDR).…”