Phosphorylation Impacts N-end Rule Degradation of the Proteolytically Activated Form of BMX Kinase

Abstract

Cellular signaling leading to the initiation of apoptosis typically results in the activation of caspases, which in turn leads to the proteolytic generation of protein fragments with new or altered cellular functions. Increasing numbers of reports are demonstrating that the activity of many of these proteolytically activated protein fragments can be attenuated by their selective degradation by the N-end rule pathway. Here we report the first evidence that selective degradation of a caspase product by the N-end rule pathway can be modulated by phosphorylation. We demonstrate that the pro-apoptotic fragment of the bone marrow kinase on chromosome X (BMX) generated by caspase cleavage in the prostate cancer-derived PC3 cell line is metabolically unstable in cells because its N-terminal tryptophan targets it for proteasomal degradation via the N-end rule pathway. In addition, we have demonstrated that phosphorylation of tyrosine 566 relatively inhibits degradation of the C-terminal BMX catalytic fragment, and this phosphorylation is crucial for its pro-apoptotic function. Overall, our results demonstrate that cleaved BMX is a novel N-end rule substrate, and its degradation exhibits a novel interplay between substrate phosphorylation and N-end rule degradation, revealing an increasing complex regulatory network of apoptotic proteolytic signaling cascades.

Emerging branches of the N-end rule pathways are revealing the sequence complexities of N-termini dependent protein degradation

“…For example, many pro-apoptotic protein fragments generated by proteolytic cleavage have been demonstrated to be targeted for degradation via the N-end rule pathway (Ditzel et al 2003;Eldeeb and Fahlman 2016a;Eldeeb and Fahlman 2016b;Piatkov et al 2012a;Piatkov et al 2014;Xu et al 2012). In addition to this, there are examples demonstrating that the inhibition of the N-end rule or knockouts of components of the N-end rule pathway sensitize cells to cell stress and cell death (Agarwalla and Banerjee 2016;Deka et al 2016;Piatkov et al 2012a).…”

“…Furthermore, it shall provide the molecular basis for a potential cellular mechanism that can regulate the degradation of a protein via post translational modifications that may be analogously identified in the future as we have previously demonstrated for an internal phosphorylation site modulating the degradation of the BMX kinase by the N-end rule pathway (Eldeeb and Fahlman 2016b). (Park et al 2015).…”

Microwave-Assisted Synthesis of Imidazo[4,5-f][1,10]phenanthroline Derivatives as Apoptosis Inducers in Chemotherapy by Stabilizing Bcl-2 G-quadruplex DNA

“…At concentration of 20 µM for 1 , the ratio of G1-phase reached about 67.37%, which was about 12% higher than that of the control (55.75%) [ 17 , 18 ]. These data indicating that 1 may inhibit the growth of A549 cells through inducing apoptosis of tumor cells since G1 phase arrest was more related to cell apoptosis, and pro-apoptotic proteins like Bcl-2 usually plays key roles in accelerating the apoptosis of tumor cells [ 19 , 20 , 21 , 22 , 23 ].…”

A Novel Role of Dickkopf-Related Protein 3 in Macropinocytosis in Human Bladder Cancer T24 Cells

“…The N-end rule pathway recognizes specific N-terminal residues and ubiquitinates the proteins for proteasomal degradation [20]. If T24 cells had a mutation or modification, including phosphorylation in the N-terminal residues of the Dkk-3 protein, degradation of the Dkk-3 protein may delay and lead to its specific overexpression in T24 cells [21]. However, our data demonstrated that Dkk-3 protein in T24 cells was degraded by the proteasome as observed in 253JB-V cells.…”