Christopher J. Clarke

Phosphorylation plays a central role in regulating signal transduction across all kingdoms of life, allowing organisms to sense and respond to their environment. In mammals, the signalling research field is dominated by the functions of pSer, pThr and pTyr, due to both historical and technological factors. Mostly ignored are the labile phosphosites (LaPhs), made up of six other phosphorylatable amino acids: His, Lys, Arg, Asp, Glu and Cys. This group is characterised by an acid and/or heat-labile phosphate linkage, forming a distinct group from the highly stable phosphomonoesters of pSer, pThr and pTyr. LaPhs have distinct thermal and pH stability profiles, which may contribute to, or even dictate, their functions. Here, we review the contribution of LaPhs to mammalian signalling networks, highlighting their currently defined diverse functions.

Protein phosphorylation is a ubiquitous post-translational modification (PTM) found across the kingdoms of life, and is critical for the regulation of protein function in health & disease. Advances in high-throughput mass spectrometry have transformed our ability to interrogate the phosphoproteome. However, sample preparation methodologies optimized for phosphoproteomics have not kept pace, compromising the ability to fully exploit these technological advances. In this study, we present an optimized phosphoproteomics workflow using carboxylated SP3 magnetic beads which have simplified proteomics sample preparation. By employing a washing step with 8 M urea and omitting the conventional C ₁₈ SPE clean-up, we demonstrate a significant improvement in phosphopeptide identifications, with application of this refined protocol to HEK-293T cell extracts increasing the number nearly 2-fold compared to standard SP3 techniques (7908 cf . 4129). We also observed a substantial improvement in the detection of multiply phosphorylated peptides. Our findings suggest that the complexity of PTM crosstalk using current peptide-based proteomics workflows is currently under-represented and underscores the necessity of methodological innovations to better capture the intricacies of the phosphoproteome landscape.

This feature article discusses mass spectrometry-based strategies for the characterisation, localisation and differentiation of phosphorylation and sulfation on proteins, and considers future directions in the field.

Polo-like kinase 4 (PLK4) is the master regulator of centriole duplication in metazoan organisms. Catalytic activity and protein turnover of PLK4 are tightly coupled in human cells, since changes in PLK4 concentration and catalysis have profound effects on centriole duplication and supernumerary centrosomes, which are associated with aneuploidy and cancer. Recently, PLK4 has been targeted with a variety of small molecule kinase inhibitors exemplified by centrinone, which rapidly induces inhibitory effects on PLK4 and leads to on-target centrosome depletion. Despite this, relatively few PLK4 substrates have been identified unequivocally in human cells, and PLK4 signaling outside centriolar networks remains poorly characterised. We report an unbiased mass spectrometry (MS)-based quantitative analysis of cellular protein phosphorylation in stable PLK4-expressing U2OS human cells exposed to centrinone. PLK4 phosphorylation was itself sensitive to brief exposure to the compound, resulting in PLK4 stabilization. Analysing asynchronous cell populations, we report hundreds of centrinone-regulated cellular phosphoproteins, including centrosomal and cell cycle proteins and a variety of likely ‘non-canonical’ substrates. Surprisingly, sequence interrogation of ~300 significantly down-regulated phosphoproteins reveals an extensive network of centrinone-sensitive [Ser/Thr]Pro phosphorylation sequence motifs, which based on our analysis might be either direct or indirect targets of PLK4. In addition, we confirm that NMYC and PTPN12 are PLK4 substrates, both in vitro and in human cells. Our findings suggest that PLK4 catalytic output directly controls the phosphorylation of a diverse set of cellular proteins, including Pro-directed targets that are likely to be important in PLK4-mediated cell signaling.