Identification of DYRK1B as a substrate of ERK1/2 and characterisation of the kinase activity of DYRK1B mutants from cancer and metabolic syndrome.

The dual-specificity tyrosine-phosphorylation-regulated kinase, DYRK1B, is expressed de novo during myogenesis, amplified or mutated in certain cancers and mutated in familial cases of metabolic syndrome. DYRK1B is activated by cis auto-phosphorylation on tyrosine-273 (Y273) within the activation loop during translation but few other DYRK1B phosphorylation sites have been characterised to date. Here, we demonstrate that DYRK1B also undergoes trans-autophosphorylation on serine-421 (S421) in vitro and in cells and that this site contributes to DYRK1B kinase activity. Whilst a DYRK1B(S421A) mutant was completely defective for p-S421 in cells, DYRK1B inhibitors caused only a partial loss of p-S421 suggesting the existence of an additional kinase that could also phosphorylate DYRK1B S421. Indeed, a catalytically inactive DYRK1B(D239A) mutant exhibited very low levels of p-S421 in cells but this was increased by KRAS(G12V). In addition, selective activation of the RAF-MEK1/2-ERK1/2 signalling pathway rapidly increased p-S421 in cells whereas activation of the stress kinases JNK or p38 could not. S421 resides within a Ser-Pro phosphoacceptor motif that is typical for ERK1/2 and recombinant ERK2 phosphorylated DYRK1B at S421 in vitro. Our results show that DYRK1B is a novel ERK2 substrate, uncovering new links between two kinases involved in cell fate decisions. Finally, we show that DYRK1B mutants that have recently been described in cancer and metabolic syndrome exhibit normal or reduced intrinsic kinase activity.

Prostatic tissue protein alterations due to delayed time to freezing.

Clinical studies often produce fresh tissue samples, which ideally should be immediately snap frozen for storage and subsequent analysis. However, this is often not practically possible, and there is inevitably a time period during which the sample is stored on ice. The delay in freezing may allow endogenous degradation of proteins to occur, affecting 2-D gel protein profiles. This study aims to investigate the type and extent of this degradation by examining how the time-to-freezing delay alters prostatic tissue protein profile. The prostate carcinoma-3 cell line (PC-3), prostate cancer xenografts and canine prostate were used with fluorescence 2-D DIGE to assess protein degradation. It was found that 30-min processing time had minimal effects on the protein profile. Longer delays had little visible effect, but subtle alterations in protein profile began to accumulate as time increased. These data support the practice of completing tissue processing as rapidly as possible, and indicate that short processing times do not notably perturb the 2-D gel spot pattern from prostatic tissue.