Tools to discriminate between targets of CK2 vs PLK2/PLK3 acidophilic kinases

While the great majority of Ser/Thr protein kinases are basophilic or proline directed, a tiny minority is acidophilic. The most striking example of such "acidophilic" kinases is CK2, whose sites are specified by numerous acidic residues surrounding the target one. However PLK2 and PLK3 kinases recognize an acidic consensus similar to CK2 when tested on peptide libraries. Here we describe optimal buffer conditions for PLK2 and 3 kinase activity assays and tools such as using GTP as a phosphate donor and the specific inhibitors CX-4945 and BI 2536, useful to discriminate between acidic phosphosites generated either by CK2 or by PLK2/PLK

Superiority of PLK-2 as \u3b1-synuclein phosphorylating agent relies on unique specificity determinants.

Phosphorylation of \u3b1-synuclein at Ser-129 is of crucial relevance to Parkinson's disease and related synucleinopathies. Here we provide biochemical evidence that PLK2 and to a lesser extent PLK3 are superior over CK2, as catalysts of Ser-129 phosphorylation both in full length \u3b1-synuclein and in a peptide reproducing the C-terminal segment of the protein. By using substituted peptides we also show that the sequence surrounding Ser-129 is optimally shaped for undergoing phosphorylation by PLK2, with special reference to the two acidic residues at positions n-3 (Glu-126) and n+2 (Glu-131) whose replacement with alanine abrogates phosphorylation

Investigation on PLK2 and PLK3 substrate recognition.

Analyses of human phosphoproteome based on primary structure of the aminoacids surrounding the phosphor Ser/Thr suggest that a significant proportion of phosphosites is generated by a restricted number of acidophilic kinases, among which protein kinase CK2 plays a prominent role. Recently, new acidophilic kinases belonging to the Polo like kinase family have been characterized, with special reference to PLK1, PLK2, and PLK3 kinases. While some progress has been made in deciphering the PLK1-dependent phosphoproteome, very little is known about the targets of PLK2 and PLK3 kinases. In this report by using an in vitro approach, consisting of cell lysate phosphorylation, phosphoprotein separation by 2D gel electrophoresis and mass spectrometry, we describe the identification of new potential substrates of PLK2 and PLK3 kinases. We have identified and validated as in vitro PLK2 and PLK3 substrates HSP90, GRP-94, \u3b2-tubulin, calumenin, and 14-3-3 epsilon. The phosphosites generated by PLK3 in these proteins have been identified by mass spectrometry analysis to get new insights about PLKs specificity determinants. These latter have been further corroborated by an in silico analysis of the PLKs substrate binding region

Carrier Gas Triggered Controlled Biolistic Delivery of DNA and Protein Therapeutics from Metal-Organic Frameworks

Abstract: The efficacy and specificity of protein, DNA, and RNA-based drugs have made them popular in the clinic; however, their susceptibility to environmental stressors adds significant challenges to formulating biomacromolecules into delivery systems where the kinetics of release can be tuned. Further, these drugs are often delivered via injection, which requires skilled medical personnel and produces biohazardous waste. Here, we report an approach that allows for the controlled delivery of DNA and protein therapeutics to allow for either burst or slow-release kinetics without altering the formulation; further, we show we can deliver these materials into the tissues of very different organisms without the use of needles. We show that biomaterials encapsulated within the highly porous metal-organic Framework ZIF-8 are stable as a powder formulation that can be shot into tissue with a low-cost gas-powered “MOF-Jet” for direct delivery into living tissues of plants and animals and the release of the biomaterials can be controlled by judiciously choosing the compressed gas used in the gun. Many MOFs, including ZIF-8, are acid labile and readily dissolve at low pH. When CO2 is used as the carrier gas to shoot MOFs into moist tissue, we show that we can create a transient and weakly acidic local environment that causes the near-instantaneous release of the biomolecules. Conversely, when air is used, the MOF is delivered into tissue and degrades slowly over a week, releasing biomolecules. This innovation represents the first example of biolistic-mediated controlled delivery of biomolecules with ZIF-8 and provides a powerful tool for fundamental and applied plant and animal sciences research