Aline Santos

Shp2 negatively regulates growth in cardiomyocytes by controlling focal adhesion kinase/Src and mTOR pathways

The aim of this study was to investigate whether Shp2 (Src homology region 2, phosphatase 2) controls focal adhesion kinase (FAK) activity and its trophic actions in cardiomyocytes. We show that low phosphorylation levels of FAK in nonstretched neonatal rat ventricular myocytes (NRVMs) coincided with a relatively high basal association of FAK with Shp2 and Shp2 phosphatase activity. Cyclic stretch (15% above initial length) enhanced FAK phosphorylation at Tyr397 and reduced FAK/Shp2 association and phosphatase activity in anti-Shp2 precipitates. Recombinant Shp2 C-terminal protein tyrosine phosphatase domain (Shp2-PTP) interacted with nonphosphorylated recombinant FAK and dephosphorylated FAK immunoprecipitated from NRVMs. Depletion of Shp2 by specific small interfering RNA increased the phosphorylation of FAK Tyr397, Src Tyr418, AKT Ser473, TSC2 Thr1462, and S6 kinase Thr389 and induced hypertrophy of nonstretched NRVMs. Inhibition of FAK/Src activity by PP2 {4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine} abolished the phosphorylation of AKT, TSC2, and S6 kinase, as well as the hypertrophy of NRVMs induced by Shp2 depletion. Inhibition of mTOR (mammalian target of rapamycin) with rapamycin blunted the hypertrophy in NRVMs depleted of Shp2. NRVMs treated with PP2 or depleted of FAK by specific small interfering RNA were defective in FAK, Src, extracellular signal-regulated kinase, AKT, TSC2, and S6 kinase phosphorylation, as well as in the hypertrophic response to prolonged stretch. The stretch-induced hypertrophy of NRVMs was also prevented by rapamycin. These findings demonstrate that basal Shp2 tyrosine phosphatase activity controls the size of cardiomyocytes by downregulating a pathway that involves FAK/Src and mTOR signaling pathways.

αB-crystallin interacts with and prevents stress-activated proteolysis of focal adhesion kinase by calpain in cardiomyocytes.

Focal adhesion kinase (FAK) contributes to cellular homeostasis under stress conditions. Here we show that αB-crystallin interacts with and confers protection to FAK against calpain-mediated proteolysis in cardiomyocytes. A hydrophobic patch mapped between helices 1 and 4 of the FAK FAT domain was found to bind to the β4-β8 groove of αB-crystallin. Such an interaction requires FAK tyrosine 925 and is enhanced following its phosphorylation by Src, which occurs upon FAK stimulation. αB-crystallin silencing results in calpain-dependent FAK depletion and in the increased apoptosis of cardiomyocytes in response to mechanical stress. FAK overexpression protects cardiomyocytes depleted of αB-crystallin against the stretch-induced apoptosis. Consistently, load-induced apoptosis is blunted in the hearts from cardiac-specific FAK transgenic mice transiently depleted of αB-crystallin by RNA interference. These studies define a role for αB-crystallin in controlling FAK function and cardiomyocyte survival through the prevention of calpain-mediated degradation of FAK.

FERM domain interaction with myosin negatively regulates FAK in cardiomyocyte hypertrophy

Focal adhesion kinase (FAK) regulates cellular processes that affect several aspects of development and disease. The FAK N-terminal FERM (4.1 protein-ezrin-radixin-moesin homology) domain, a compact clover-leaf structure, binds partner proteins and mediates intramolecular regulatory interactions. Combined chemical cross-linking coupled to MS, small-angle X-ray scattering, computational docking and mutational analyses showed that the FAK FERM domain has a molecular cleft (~998 Å(2)) that interacts with sarcomeric myosin, resulting in FAK inhibition. Accordingly, mutations in a unique short amino acid sequence of the FERM myosin cleft, FP-1, impaired the interaction with myosin and enhanced FAK activity in cardiomyocytes. An FP-1 decoy peptide selectively inhibited myosin interaction and increased FAK activity, promoting cardiomyocyte hypertrophy through activation of the AKT-mammalian target of rapamycin pathway. Our findings uncover an inhibitory interaction between the FAK FERM domain and sarcomeric myosin that presents potential opportunities to modulate the cardiac hypertrophic response through changes in FAK activity.

Analysis of secondary structure in proteins by chemical cross-linking coupled to MS

Chemical cross‐linking is an attractive technique for the study of the structure of protein complexes due to its low sample consumption and short analysis time. Furthermore, distance constraints obtained from the identification of cross‐linked peptides by MS can be used to construct and validate protein models. If a sufficient number of distance constraints are obtained, then determining the secondary structure of a protein can allow inference of the proteins fold. In this work, we show how the distance constraints obtained from cross‐linking experiments can identify secondary structures within the protein sequence. Molecular modeling of alpha helices and beta sheets reveals that each secondary structure presents different cross‐linking possibilities due to the topological distances between reactive residues. Cross‐linking experiments performed with amine reactive cross‐linkers with model alpha helix containing proteins corroborated the molecular modeling predictions. The cross‐linking patterns established here can be extended to other cross‐linkers with known lengths for the determination of secondary structures in proteins.

αB‐Crystallin interacts and attenuates the tyrosine phosphatase activity of Shp2 in cardiomyocytes under mechanical stress

The small heat shock protein αB‐Crystallin (CryAB, HspB5) and SH2 domain‐containing tyrosine phosphatase 2 (Shp2) are important molecules in heart response to pathophysiological stress. Here we show that CryAB interacts with and potentially regulates Shp2 catalytic activity in stretched cardiomyocytes. Such an interaction requires CryAB oligomer to attenuate Shp2 activation. Stretched cardiomyocytes show a robust CryAB/Shp2 association accompanied by a reduction in the Shp2 phosphatase activity. Accordingly, CryAB knock‐down in cardiomyocytes enhances Shp2 activity induced by mechanical stress. These results revealed a new role for CryAB, as a modulator of Shp2 phosphatase activity during a functionally relevant stimulus in cardiomyocytes.

Intraspecific variability of popcorn S7 lines for phosphorus efficiency in the soil

The expansion of agriculture, coupled with the need for sustainable cropping, is one of the greatest challenges of the scientific community working on the generation of new cultivars adapted to abiotic stress conditions. The aim of this study was to evaluate the variability of popcorn lines as to responsiveness and efficiency in phosphorus use, as a first step towards the implementation of a breeding program interested in the practice of sustainable agriculture. Twenty-five popcorn lines were evaluated in two locations with different phosphorus levels in the soil, using a randomized block design. The following traits were measured: plant height, ear height, female flowering date, male flowering date, male-female flowering interval, ear diameter, ear length, 100-grain weight, grain yield, popping expansion, and expanded popcorn volume per hectare. A combined analysis of variance and test of means were performed, and the lines were classified as to their phosphorus use efficiency, according to their production performance in the different environments. The genetic diversity between the lines was estimated by Tochers and UPGMA clustering methods, using generalized Mahalanobis distance. Lines L59, P7, P2, P3, P4, P8, P10, P9, L66, L70, L69, and P5 were efficient and responsive, whereas lines L75, L80, L61, L77, L63, L65, P1, L54, L53, L88, and L71 were inefficient and nonresponsive. Genetic variability was greater in the environments with low phosphorus in the soil, suggesting that the selection pressure exerted in the stressing environment is a decisive factor to obtain a higher expression of variability.