Rabbit skeletal muscle myosin light chain kinase. The calmodulin binding domain as a potential active site-directed inhibitory domain.

Journal ArticleA synthetic peptide modeled after the calmodulin (CaM)-binding domain of rabbit skeletal muscle myosin light chain kinase, Lys-Arg-Arg-Trp-Lys5-Lys-Asn-Phe-Ile-Ala10-Val-Ser-Ala-Ala-+ ++Asn15-Arg-Phe-Glycyl amide (M5), inhibited the CaM-independent chymotryptic fragment of the enzyme, C35 (Edelman, A. M., Takio, K., Blumenthal, D. K., Hansen, R. S., Walsh, K. A., Titani, K., and Krebs, E. G. (1985) J. Biol. Chem. 260, 11275-11285), with a Ki of 3.2 +/- 2.1 microM. Inhibition was competitive with respect to the peptide substrate Lys-Lys-Arg-Ala-Ala5-Arg-Ala-Thr-Ser-Asn10-Val-Phe-Ala and was of the noncompetitive linear mixed type with respect to ATP. M5 and homologues with a serine residue substituted at positions 9, 13, or 14 were phosphorylated with the following order of preference: M5(Ser9) greater than M5(Ser13) much greater than M5(Ser14) greater than M5. The order of preference observed agreed with that predicted by comparison of the sequence of these peptides with the phosphorylation sites of myosin P-light chains. Both inhibition of C35 by M5 and phosphorylation of M5 and its serine-substituted homologues were severely curtailed by the addition of a stoichiometric excess of CaM over peptide. Thus, synthetic peptides modeled after the CaM-binding domain of skeletal muscle myosin light chain kinase can function as calmodulin-regulated active site-directed inhibitors of the enzyme

Protein Tyrosine Phosphorylation in the Cyanobacterium Anabaena PCC 7120

Components of a protein tyrosine phosphorylation/dephosphorylation network were identified in the cyanobacterium Anabaena sp. strain PCC 7120. Three phosphotyrosine (P-Tyr) proteins of 27, 36, and 52 kDa were identified through their conspicuous immunoreactions with RC20H monoclonal antibodies specific for P-Tyr. These immunoreactions were outcompeted completely by free P-Tyr (5 mM) but not by phosphoserine or phosphothreonine. The P-Tyr content of the three major P-Tyr proteins and several minor proteins increased with their time of incubation in the presence of Mg-ATP and the protein phosphatase inhibitors sodium orthovanadate and sodium fluoride. Incubation of the same extracts with [g-32P]ATP but not [a-32P]ATP led to the phosphorylation of five polypeptides with molecular masses of 20, 27, 52, 85, and 100 kDa. Human placental protein tyrosine phosphatase 1B, with absolute specificity for P-Tyr, liberated significant quantities of 32Pi from four of the polypeptides, confirming that a portion of the protein-bound phosphate was present as 32P-Tyr. Alkaline phosphatase and the dual-specificity protein phosphatase IphP from the cyanobacterium Nostoc commune UTEX 584 also dephosphorylated these proteins and did so with greater apparent efficiency. Two of the polypeptides were partially purified, and phosphoamino analysis identified 32P-Tyr, [32P]phosphoserine, and [32P]phosphothreonine. Anabaena sp. strain PCC 7120 cell extracts contained a protein tyrosine phosphatase activity that was abolished in the presence of sodium orthovanadate and inhibited significantly by the sulfhydryl-modifying agents p-hydroxymercuriphenylsulfonic acid and p-hydroxymercuribenzoate as well as by heparin. In Anabaena sp. strain PCC 7120 the presence and/or phosphorylation status of P-Tyr proteins was influenced by incident photon flux density

Activation mechanism of rabbit skeletal muscle myosin light chain kinase 5′-p-Fluorosulfonylbenzoyl adenosine as a probe of the MgATP-binding site of the calmodulin-bound and calmodulin-free enzyme

Abstract5′-p-Fluorosulfonylbenzoyl adenosine (FSBA), an ATP-like affinity labelling reagent, reacted with rabbit skeletal muscle myosin light chain kinase (skMLCK) and its calmodulin complex in a site-specific manner. Reaction was dependent upon the presence of the adenosine moiety of FSBA, saturated with increasing FSBA, was inhibited by MgATP, and was accompanied by stoichiometric incorporation of [14C]FSBA. The kinetic constants describing the reaction were similar for skMLCK and its calmodulin complex: k3= −0.040 min−1 and −0.038 mint-1, and Ki=0.18 mM and 0.40 mM, respectively. It is concluded that the MgATP-binding site on skMLCK remains accessible at all times and maintains a near constant conformation

A Planet Orbiting the Star Rho Coronae Borealis

We report the discovery of near-sinusoidal radial velocity variations of the G0V star rhoCrB, with period 39.6 days and amplitude 67 m/s. These variations are consistent with the existence of an orbital companion in a circular orbit. Adopting a mass of 1.0 M(Sun) for the primary, the companion has minimum mass about 1.1 Jupiter masses, and orbital radius about 0.23 AU. Such an orbital radius is too large for tidal circularization of an initially eccentric orbit during the lifetime of the star, and hence we suggest that the low eccentricity is primordial, as would be expected for a planet formed in a dissipative circumstellar disk.Comment: 9 pages, LaTeX, accepted in Astrophys. J. Letter

Ser/Thr/Tyr Protein Phosphorylation in the Archaeon Halobacterium salinarum—A Representative of the Third Domain of Life

In the quest for the origin and evolution of protein phosphorylation, the major regulatory post-translational modification in eukaryotes, the members of archaea, the “third domain of life”, play a protagonistic role. A plethora of studies have demonstrated that archaeal proteins are subject to post-translational modification by covalent phosphorylation, but little is known concerning the identities of the proteins affected, the impact on their functionality, the physiological roles of archaeal protein phosphorylation/dephosphorylation, and the protein kinases/phosphatases involved. These limited studies led to the initial hypothesis that archaea, similarly to other prokaryotes, use mainly histidine/aspartate phosphorylation, in their two-component systems representing a paradigm of prokaryotic signal transduction, while eukaryotes mostly use Ser/Thr/Tyr phosphorylation for creating highly sophisticated regulatory networks. In antithesis to the above hypothesis, several studies showed that Ser/Thr/Tyr phosphorylation is also common in the bacterial cell, and here we present the first genome-wide phosphoproteomic analysis of the model organism of archaea, Halobacterium salinarum, proving the existence/conservation of Ser/Thr/Tyr phosphorylation in the “third domain” of life, allowing a better understanding of the origin and evolution of the so-called “Nature's premier” mechanism for regulating the functional properties of proteins

The impact of surgical delay on resectability of colorectal cancer: An international prospective cohort study

AIM: The SARS-CoV-2 pandemic has provided a unique opportunity to explore the impact of surgical delays on cancer resectability. This study aimed to compare resectability for colorectal cancer patients undergoing delayed versus non-delayed surgery. METHODS: This was an international prospective cohort study of consecutive colorectal cancer patients with a decision for curative surgery (January-April 2020). Surgical delay was defined as an operation taking place more than 4 weeks after treatment decision, in a patient who did not receive neoadjuvant therapy. A subgroup analysis explored the effects of delay in elective patients only. The impact of longer delays was explored in a sensitivity analysis. The primary outcome was complete resection, defined as curative resection with an R0 margin. RESULTS: Overall, 5453 patients from 304 hospitals in 47 countries were included, of whom 6.6% (358/5453) did not receive their planned operation. Of the 4304 operated patients without neoadjuvant therapy, 40.5% (1744/4304) were delayed beyond 4 weeks. Delayed patients were more likely to be older, men, more comorbid, have higher body mass index and have rectal cancer and early stage disease. Delayed patients had higher unadjusted rates of complete resection (93.7% vs. 91.9%, P = 0.032) and lower rates of emergency surgery (4.5% vs. 22.5%, P < 0.001). After adjustment, delay was not associated with a lower rate of complete resection (OR 1.18, 95% CI 0.90-1.55, P = 0.224), which was consistent in elective patients only (OR 0.94, 95% CI 0.69-1.27, P = 0.672). Longer delays were not associated with poorer outcomes. CONCLUSION: One in 15 colorectal cancer patients did not receive their planned operation during the first wave of COVID-19. Surgical delay did not appear to compromise resectability, raising the hypothesis that any reduction in long-term survival attributable to delays is likely to be due to micro-metastatic disease

Archaeal protein kinases and protein phosphatases: insights from genomics and biochemistry.

Protein phosphorylation/dephosphorylation has long been considered a recent addition to Nature's regulatory arsenal. Early studies indicated that this molecular regulatory mechanism existed only in higher eukaryotes, suggesting that protein phosphorylation/dephosphorylation had emerged to meet the particular signal-transduction requirements of multicellular organisms. Although it has since become apparent that simple eukaryotes and even bacteria are sites of protein phosphorylation/dephosphorylation, the perception widely persists that this molecular regulatory mechanism emerged late in evolution, i.e. after the divergence of the contemporary phylogenetic domains. Only highly developed cells, it was reasoned, could afford the high 'overhead' costs inherent in the acquisition of dedicated protein kinases and protein phosphatases. The advent of genome sequencing has provided an opportunity to exploit Nature's phylogenetic diversity as a vehicle for critically examining this hypothesis. In tracing the origins and evolution of protein phosphorylation/dephosphorylation, the members of the Archaea, the so-called 'third domain of life', will play a critical role. Whereas several studies have demonstrated that archaeal proteins are subject to modification by covalent phosphorylation, relatively little is known concerning the identities of the proteins affected, the impact on their functional properties, or the enzymes that catalyse these events. However, examination of several archaeal genomes has revealed the widespread presence of several ostensibly 'eukaryotic' and 'bacterial' protein kinase and protein phosphatase paradigms. Similar findings of 'phylogenetic trespass' in members of the Eucarya (eukaryotes) and the Bacteria suggest that this versatile molecular regulatory mechanism emerged at an unexpectedly early point in development of 'life as we know it'