Phosphorylation of telokin by cyclic nucleotide kinases and the identification of in vivo phosphorylation sites in smooth muscle

AbstractThe Ca2+-independent acceleration of dephosphorylation of the regulatory light chain of smooth muscle myosin and relaxation of smooth muscle by telokin are enhanced by cyclic nucleotide-activated protein kinase(s) [Wu et al. (1998) J. Biol. Chem. 273, 11362–11369]. The purpose of this study was to determine the in vivo site(s) and in vitro rates of telokin phosphorylation and to evaluate the possible effects of sequential phosphorylation by different kinases. The in vivo site(s) of phosphorylation of telokin were determined in rabbit smooth muscles of longitudinal ileum and portal vein. Following stimulation of ileum with forskolin (20 μM) the serine at position 13 was the only amino acid to exhibit increased phosphorylation. Rabbit portal vein telokin was phosphorylated on both Ser-13 and -19 as a result of forskolin and GTPγS stimulation in vivo. Point mutation of Ser-13 (to Ala or Asp) abolished in vitro phosphorylation by cyclic nucleotide-dependent protein kinases

Identification of an Allosteric Small-Molecule Inhibitor Selective for the Inducible Form of Heat Shock Protein 70

Inducible Hsp70 (Hsp70i) is overexpressed in a wide spectrum of human tumors and its expression correlates with metastasis, poor outcomes, and resistance to chemotherapy in patients. Identification of small molecule inhibitors selective for Hsp70i could provide new therapeutic tools for cancer treatment. In this work, we used fluorescence-linked enzyme chemoproteomic strategy (FLECS) to identify HS-72, an allosteric inhibitor selective for Hsp70i. HS-72 displays the hallmarks of Hsp70 inhibition in cells, promoting substrate protein degradation and growth inhibition. Importantly, HS-72 is selective for Hsp70i over the closely related constitutively active Hsc70. Studies with purified protein show HS-72 acts as an allosteric inhibitor, reducing ATP affinity. In vivo HS-72 is well-tolerated, showing bioavailability and efficacy, inhibiting tumor growth and promoting survival in a HER2+ model of breast cancer. The HS-72 scaffold is amenable to resynthesis and iteration, suggesting an ideal starting point for a new generation of anticancer therapeutics targeting Hsp70i

Modulation of smooth muscle contractility by CHASM, a novel member of the smoothelin family of proteins

AbstractCyclic nucleotides acting through their associated protein kinases, the cGMP- and cAMP-dependent protein kinases, can relax smooth muscles without a change in free intracellular calcium concentration ([Ca2+]i), a phenomenon referred to as Ca2+ desensitization. The molecular mechanisms by which these kinases bring about Ca2+ desensitization are unknown and an understanding of this phenomenon may lead to better therapies for treating diseases involving defects in the contractile response of smooth muscles such as hypertension, bronchospasm, sexual dysfunction, gastrointestinal disorders and glaucoma. Utilizing a combination of real-time proteomics and smooth muscle physiology, we characterized a distinct subset of protein targets for cGMP-dependent protein kinase in smooth muscle. Among those phosphoproteins identified was calponin homology-associated smooth muscle (CHASM), a novel protein that contains a calponin homology domain and shares sequence similarity with the smoothelin family of smooth muscle specific proteins. Recombinant CHASM was found to evoke relaxation in a concentration dependent manner when added to permeabilized smooth muscle. A co-sedimentation assay with actin demonstrated that CHASM does not possess actin binding activity. Our findings indicate that CHASM is a novel member of the smoothelin protein family that elicits Ca2+ desensitization in smooth muscle

Roles of the AMP‐activated and cyclic‐AMP‐dependent protein kinases in the adrenaline‐induced inactivation of acetyl‐CoA carboxylase in rat adipocytes

In isolated rat adipocytes, acetyl‐CoA carboxylase is inactivated by treatment of the cells with adrenaline or the β‐agonist isoproterenol, but not by the α‐agonist phenylephine. The inactivation is stable during purification in the presence of protein phosphatase inhibitors, and is associated with a 30–40% increase in the labelling of enzyme isolated from 32P‐labelled cells. Increased phosphorylation occurs within peptide T1, which was identified by sequencing to be the peptide Ser‐Ser77‐Met‐Ser79‐Gly‐Leu‐His‐Leu‐Val‐Lys, containing Ser‐77 (phosphorylated by cyclic‐AMP‐dependent protein kinase) and Ser‐79 (phosphorylated by the AMP‐activated protein kinase). Analysis of the release of radioactivity as free phosphate during Edman degradation of peptide T1 revealed that all of the phosphate was in Ser‐79 in both basal and hormone‐ or agonist‐stimulated cells. Treatment of adipocytes with various agents which activate cyclic‐AMP‐dependent protein kinase by receptor‐independent mechanisms (forskolin, cyclic AMP analogues, isobutylmethylxanthine) also produced inactivation of acetyl‐CoA carboxylase and increased phosphorylation at Ser‐79. The (Rp)‐[thio]phosphate analogue of cyclic AMP, which is an antagonist of binding of cyclic AMP to the regulatory subunit of cyclic‐AMP‐dependent protein kinase, opposes the effect of adrenaline on phosphorylation and inactivation of acetyl‐CoA carboxylase. Together with the effects of isobutylmethylxanthine and the stimulatory cyclic AMP analogues, this strongly indicates that cyclic‐AMP‐dependent protein kinase is an essential component of the signal transduction pathway, although clearly it does not directly phosphorylate acetyl‐CoA carboxylase. As shown by okadaic acid inhibition, &gt; 95% of the acetyl‐CoA carboxylase phosphatase activity in extracts of rat adipocytes or liver is accounted for by protein phosphatase‐2A, with &lt;5% attributable to protein phosphatase‐1. Inhibition of protein phosphatase‐1 via phosphorylation of inhibitor‐1 is therefore unlikely to be the mechanism by which cyclic‐AMP‐dependent protein kinase indirectly increases phosphorylation of acetyl‐CoA carboxylase. Various other potential mechanisms are discussed.</p

Inhibition of Both Hsp70 Activity and Tau Aggregation in \u3cem\u3eVitro\u3c/em\u3e Best Predicts Tau Lowering Activity of Small Molecules

Three scaffolds with inhibitory activity against the heat shock protein 70 (Hsp70) family of chaperones have been found to enhance the degradation of the microtubule associated protein tau in cells, neurons, and brain tissue. This is important because tau accumulation is linked to neurodegenerative diseases including Alzheimer’s disease (AD) and chronic traumatic encephalopathy (CTE). Here, we expanded upon this study to investigate the anti-tau efficacy of additional scaffolds with Hsp70 inhibitory activity. Five of the nine scaffolds tested lowered tau levels, with the rhodacyanine and phenothiazine scaffolds exhibiting the highest potency as previously described. Because phenothiazines also inhibit tau aggregation in vitro, we suspected that this activity might be a more accurate predictor of tau lowering. Interestingly, the rhodacyanines did inhibit in vitro tau aggregation to a similar degree as phenothiazines, correlating well with tau-lowering efficacy in cells and ex vivo slices. Moreover, other Hsp70 inhibitor scaffolds with weaker tau-lowering activity in cells inhibited tau aggregation in vitro, albeit at lower potencies. When we tested six well-characterized tau aggregation inhibitors, we determined that this mechanism of action was not a better predictor of tau-lowering than Hsp70 inhibition. Instead, we found that compounds possessing both activities were the most effective at promoting tau clearance. Moreover, cytotoxicity and PAINS activity are critical factors that can lead to false-positive lead identification. Strategies designed around these principles will likely yield more efficacious tau-lowering compounds