The Participation of Calponin in the Cross Talk between 20-Hydroxyecdysone and Juvenile Hormone Signaling Pathways by Phosphorylation Variation

20-hydroxyecdysone (20E) and juvenile hormone (JH) signaling pathways interact to mediate insect development, but the mechanism of this interaction is poorly understood. Here, a calponin homologue domain (Chd) containing protein (HaCal) is reported to play a key role in the cross talk between 20E and JH signaling by varying its phosphorylation. Chd is known as an actin binding domain present in many proteins including some signaling proteins. Using an epidermal cell line (HaEpi), HaCal was found to be up-regulated by either 20E or the JH analog methoprene (JHA). 20E induced rapid phosphorylation of HaCal whereas no phosphorylation occurred with JHA. HaCal could be quickly translocated into the nuclei through 20E or JH signaling but interacted with USP1 only under the mediation of JHA. Knockdown of HaCal by RNAi blocked the 20E inducibility of USP1, PKC and HR3, and also blocked the JHA inducibility of USP1, PKC and JHi. After gene silencing of HaCal by ingestion of dsHaCal expressed by Escherichia coli, the larval development was arrested and the gene expression of USP1, PKC, HR3 and JHi were blocked. These composite data suggest that HaCal plays roles in hormonal signaling by quickly transferring into nucleus to function as a phosphorylated form in the 20E pathway and as a non-phosphorylated form interacting with USP1 in the JH pathway to facilitate 20E or JH signaling cascade, in short, by switching its phosphorylation status to regulate insect development

Smooth Muscle Archvillin Is an ERK Scaffolding Protein*

ERK influences a number of pathways in all cells, but how ERK activities are segregated between different pathways has not been entirely clear. Using immunoprecipitation and pulldown experiments with domain-specific recombinant fragments, we show that smooth muscle archvillin (SmAV) binds ERK and members of the ERK signaling cascade in a domain-specific, stimulus-dependent, and pathway-specific manner. MEK binds specifically to the first 445 residues of SmAV. B-Raf, an upstream regulator of MEK, constitutively interacts with residues 1–445 and 446–1250. Both ERK and 14-3-3 bind to both fragments, but in a stimulus-specific manner. Phosphorylated ERK is associated only with residues 1–445. An ERK phosphorylation site was determined by mass spectrometry to reside at Ser132. A phospho-antibody raised to this site shows that the site is phosphorylated during α-agonist-mediated ERK activation in smooth muscle tissue. Phosphorylation of SmAV by ERK decreases the association of phospho-ERK with SmAV. These results, combined with previous observations, indicate that SmAV serves as a new ERK scaffolding protein and provide a mechanism for regulation of ERK binding, activation, and release from the signaling complex

Mechanism of action of angiotensin II in human isolated subcutaneous resistance arteries

1. Human isolated subcutaneous arteries were mounted in a myograph and isometric tension measured. In some experiments, intracellular calcium [Ca(2+)](i) was also measured using fura-2. 2. Angiotensin II (100 pM – 1 μM) increased [Ca(2+)](i) and tone in a concentration-dependent manner. The effects of angiotensin II (100 nM) were inhibited by an AT(1)-receptor antagonist, candesartan (100 pM). 3. Ryanodine (10 μM), had no effect on angiotensin II-induced responses, but removal of extracellular Ca(2+) abolished angiotensin II-induced rise in [Ca(2+)](i) and tone. Inhibition of Ca(2+) entry by Ni(2+) (2 mM), also inhibited angiotensin II responses. The dihydropyridine, L-type calcium channel antagonist, amlodipine (10 μM), only partially attenuated angiotensin II responses. 4. Inhibition of protein kinase C (PKC) by chelerythrine (1 μM), or by overnight exposure to a phorbol ester (PDBu; 500 nM) had no effect on angiotensin II-induced contraction. 5. Genistein (10 μM), a tyrosine kinase inhibitor, inhibited angiotensin II-induced contraction, but did not inhibit the rise in [Ca(2+)](i), suggesting that at this concentration it affected the calcium sensitivity of the contractile apparatus. Genistein did not affect responses to norepinephrine (NE) or high potassium (KPSS). 6. A selective MEK inhibitor, PD98059 (30 μM), inhibited both the angiotensin II-induced contraction and rise in [Ca(2+)](i), but had no effect on responses to NE or KPSS. 7. AT(1) activation causes Ca(2+) influx via L-type calcium channels and a dihydropyridine-insensitive route, but does not release Ca(2+) from intracellular sites. Activation of tyrosine kinase(s) and the ERK 1/2 pathway, but not classical or novel PKC, also play a role in angiotensin II-induced contraction in human subcutaneous resistance arteries