Neuronal Cdc2-like Kinase (Nclk) Binds and Phosphorylates the Retinoblastoma Protein

Abstract

The tumor suppressor retinoblastoma protein (RB) plays a central role in cellular growth regulation, differentiation, and apoptosis. Phosphorylation of RB results in a consequent loss of its ability to inhibit cell cycle progression. However, how RB phosphorylation might be regulated in apoptotic or postmitotic cells, such as neurons, remains unclear. Here we report that neuronal Cdc2-like kinase (Nclk), composed of Cdk5 and a neuronal Cdk5 activator (p25(nck5a)), can bind and phosphorylate RB. Since RB has been shown recently to associate with D-type G1 cyclins and viral oncoproteins through a common peptide sequence motif of LXCXE, Nclk binding may be mediated by a related sequence motif (LXCXXE) found in p25(nck5a). We demonstrate (i) in vitro binding of bacterially expressed p25(nck5a) to a GST-RB fusion protein, (ii) coprecipitation of GST-RB and reconstituted Cdk5.p25(nck5a), and (iii) phosphorylation of GST-RB by bacterially expressed Cdk5.p25(nck5a) kinase and by Cdk5.p25(nck5a) kinase purified from bovine brain. Finally, we show that immunoprecipitation of RB from embryonic mouse brain homogenate results in the coprecipitation of Cdk5 and that Cdk5 kinase activity is maximal during late embryonic development, a period when programmed cell death of developing neurons is greatest. Taken together, these results suggest that Nclk can bind to and phosphorylate RB in vitro and in vivo. We infer that Nclk may play an important role in regulating the activity of RB in the brain, including perhaps in apoptosing neurons.