p25/Cdk5-mediated retinoblastoma phosphorylation is an early event in neuronal cell death

Abstract

In large models of neuronal cell death, there is a tight correlation between Cdk5 deregulation and cell-cycle dysfunction. However, pathways that link Cdk5 to the cell cycle during neuronal death are still unclear. We have investigated the molecular events that precede p25/Cdk5-triggered neuronal death using a neuronal cell line that allows inducible p25 expression. In this system, no sign of apoptosis was seen before 24 hours of p25 induction. Thus, at that time, cell-cycle-regulatory proteins were analysed by immunoblotting and some of them showed a significant deregulation. Interestingly, after time-course experiments, the earliest feature correlated with p25 expression was the phosphorylation of the retinoblastoma protein (Rb). Indeed, this phosphorylation was observed 6 hours after p25 induction and was abolished in the presence of a Cdk5 inhibitor, roscovitine, which does not inhibit the usual Rb cyclin-D kinases Cdk4 and Cdk6. Furthermore, analyses of levels and subcellular localization of Cdk-related cyclins did not reveal any change following Cdk5 activation, arguing for a direct effect of Cdk5 activity on Rb protein. This latter result was clearly demonstrated by in vitro kinase assays showing that the p25-Cdk5 complex in our cell system phosphorylates Rb directly without the need for any intermediary kinase activity. Hence, Rb might be an appropriate candidate that connects Cdk5 to cell-cycle deregulation during neuronal cell death.

Neurodegeneration and Neuroinflammation in cdk5/p25-Inducible Mice

“…This delocalization can be ascribed to the fact that p25 lacks the N-terminal membrane-tethering domain of p35 (see the introduction). 5 Finally, indications for cell-cycle re-entry claimed in p25 neuroblastoma cells 46 were also evident in this novel model by increased phosphorylation of the retinoblastoma protein (Figure 1, F and G). Nevertheless, of several other and typical cell-cycle markers that we have tested, none was markedly increased in p25-expressing neurons in vivo, and this line of analysis was not further pursued.…”

RNAi screen of the druggable genome identifies modulators of proteasome inhibitor sensitivity in myeloma including CDK5

“…[28][29][30]34 CDK5's targets include several important proteins for cell growth and death, such as STAT3, AKT, BCL-2, p53, and RB. 29,[34][35][36][37] By testing multiple CDK5 siRNAs and shRNAs, we confirmed that CDK5 knockdown sensitizes genetically variable myeloma cell lines to bortezomib and other proteasome inhibitors. This finding and its clinical relevance were further explored by the use of CDK5 inhibitors, roscovitine and SCH727965, which were demonstrated to synergize with bortezomib to induce cytotoxicity of both MM cell lines and primary MM samples.…”

Kindlin-2 regulates renal tubular cell plasticity by activation of Ras and its downstream signaling

“…Immune complexes were washed three times with RIPA buffer and separated by SDS-PAGE gels. Transfer membranes were probed with indicated primary antibodies and HRP-conjugated antibody TrueBlot (eBioscience, San Diego, CA) as the secondary antibody (21). The membranes were detected by ECL, as mentioned above.…”