De novo design of a reversible phosphorylation-dependent switch for membrane targeting

Modules that switch protein-protein interactions on and off are essential to develop synthetic biology; for example, to construct orthogonal signaling pathways, to control artificial protein structures dynamically, and for protein localization in cells or protocells. In nature, the E. coli MinCDE system couples nucleotide-dependent switching of MinD dimerization to membrane targeting to trigger spatiotemporal pattern formation. Here we present a de novo peptide-based molecular switch that toggles reversibly between monomer and dimer in response to phosphorylation and dephosphorylation. In combination with other modules, we construct fusion proteins that couple switching to lipid-membrane targeting by: (i) tethering a ‘cargo’ molecule reversibly to a permanent membrane ‘anchor’; and (ii) creating a ‘membrane-avidity switch’ that mimics the MinD system but operates by reversible phosphorylation. These minimal, de novo molecular switches have potential applications for introducing dynamic processes into designed and engineered proteins to augment functions in living cells and add functionality to protocells.

Reversible detection of phosphorylation and dephosphorylation by tip-enhanced Raman spectroscopy using a cyclopentadienyl ruthenium nanotag functionalized tip

We employed a TERS tip and SERS nanotags to create a cell signaling based sensing system that is capable of creating a reversible phosphorylation/de-phosphorylation cycle for TERS measurement.

Inhibitors of Serine/Threonine Protein Phosphatases: Biochemical and Structural Studies Provide Insight for Further Development

Background:The reversible phosphorylation of proteins regulates many key functions in eukaryotic cells. Phosphorylation is catalyzed by protein kinases, with the majority of phosphorylation occurring on side chains of serine and threonine residues. The phosphomonoesters generated by protein kinases are hydrolyzed by protein phosphatases. In the absence of a phosphatase, the half-time for the hydrolysis of alkyl phosphate dianions at 25º C is over 1 trillion years; knon ~2 x 10-20 sec-1. Therefore, ser/thr phosphatases are critical for processes controlled by reversible phosphorylation.Methods:This review is based on the literature searched in available databases. We compare the catalytic mechanism of PPP-family phosphatases (PPPases) and the interactions of inhibitors that target these enzymes.Results:PPPases are metal-dependent hydrolases that enhance the rate of hydrolysis ([kcat/kM]/knon ) by a factor of ~1021, placing them among the most powerful known catalysts on earth. Biochemical and structural studies indicate that the remarkable catalytic proficiencies of PPPases are achieved by 10 conserved amino acids, DXH(X)~26DXXDR(X)~20- 26NH(X)~50H(X)~25-45R(X)~30-40H. Six act as metal-coordinating residues. Four position and orient the substrate phosphate. Together, two metal ions and the 10 catalytic residues position the phosphoryl group and an activated bridging water/hydroxide nucleophile for an inline attack upon the substrate phosphorous atom. The PPPases are conserved among species, and many structurally diverse natural toxins co-evolved to target these enzymes.Conclusion:Although the catalytic site is conserved, opportunities for the development of selective inhibitors of this important group of metalloenzymes exist.

Plant Phytochromes and their Phosphorylation

Extensive research over several decades in plant light signaling mediated by photoreceptors has identified the molecular mechanisms for how phytochromes regulate photomorphogenic development, which includes degradation of phytochrome-interacting factors (PIFs) and inactivation of COP1-SPA complexes with the accumulation of master transcription factors for photomorphogenesis, such as HY5. However, the initial biochemical mechanism for the function of phytochromes has not been fully elucidated. Plant phytochromes have long been known as phosphoproteins, and a few protein phosphatases that directly interact with and dephosphorylate phytochromes have been identified. However, there is no report thus far of a protein kinase that acts on phytochromes. On the other hand, plant phytochromes have been suggested as autophosphorylating serine/threonine protein kinases, proposing that the kinase activity might be important for their functions. Indeed, the autophosphorylation of phytochromes has been reported to play an important role in the regulation of plant light signaling. More recently, evidence that phytochromes function as protein kinases in plant light signaling has been provided using phytochrome mutants displaying reduced kinase activities. In this review, we highlight recent advances in the reversible phosphorylation of phytochromes and their functions as protein kinases in plant light signaling.

Reversible phosphorylation: a birthday tribute to Herb Tabor

Herb Tabor was the Editor-in-Chief of the Journal of Biological Chemistry (JBC) spanning the years 1971–2010. This year, Herb turns 100. What do you give a person turning 100? Our answer to this question was to dedicate two of our favorite JBC papers to Herb. Both of these papers focus on reversible phosphorylation, which we briefly review. In addition, we delve into a new finding that centers around a novel family of secreted kinases, suggesting that there are many new and exciting discoveries yet to explore.

Molecular interaction studies on ellagic acid for its anticancer potential targeting pyruvate dehydrogenase kinase 3

PDK3 plays a central role in cancer through the reversible phosphorylation of PDC thereby blocking the entry of pyruvate into the TCA cycle. PDK3 mediated metabolic switching can be therapeutically targeted for glycolysis addicted cancers.