Unique role of dystroglycan in peripheral nerve myelination,nodal structure,and sodium channel stabilization

Dystroglycan is a central component of the dystrophin-glycoprotein complex implicated in the pathogenesis of several neuromuscular diseases. Although dystroglycan is expressed by Schwann cells, its normal peripheral nerve functions are unknown. Here we show that selective deletion of Schwann cell dystroglycan results in slowed nerve conduction and nodal changes including reduced sodium channel density and disorganized microvilli. Additional features of mutant mice include deficits in rotorod performance, aberrant pain responses, and abnormal myelin sheath folding. These data indicate that dystroglycan is crucial for both myelination and nodal architecture. Dystroglycan may be required for the normal maintenance of voltage-gated sodium channels at nodes of Ranvier, possibly by mediating trans interactions between Schwann cell microvilli and the nodal axolemma.     

Dual regulated RNA transport pathways to the cortical region in developing rice endosperm

Prolamine and glutelin RNAs are localized to two subdomains of the cortical endoplasmic reticulum (ER), the protein body ER and the cisternal ER, in developing rice seeds. The addition of nearly full-length prolamine sequences at the 3' untranslated region of a reporter RNA redirects its localization from the cisternal ER to the protein body ER. Deletion analysis of prolamine RNA sequences indicates the presence of two partially redundant cis elements required for protein body ER targeting. The addition of glutelin 3' untranslated region to protein body ER cis sequences, however, redirects RNA localization to the cisternal ER. These results indicate that there are at least two regulated RNA transport pathways as well as a constitutive pathway to the cortical ER.     

Protein kinase C-epsilon-null mice have decreased hypoxic pulmonary vasoconstriction

PKC contributes to regulation of pulmonary vascular reactivity in response to hypoxia. The role of individual PKC isozymes is less clear. We used a knockout (null, -/-) mouse to test the hypothesis that PKC-epsilon is important in acute hypoxic pulmonary vasoconstriction (HPV). We asked whether deletion of PKC-epsilon would decrease acute HPV in adult C57BL6xSV129 mice. In isolated, salt solution-perfused lung, reactivity to acute hypoxic challenges (0% and 3% O(2)) was compared with responses to angiotensin II (ANG II) and KCl. PKC-epsilon -/- mice had decreased HPV, whereas responses to ANG II and KCl were preserved. Inhibition of nitric oxide synthase (NOS) with nitro-l-arginine augmented HPV in PKC-epsilon +/+ but not -/- mice. Inhibition of Ca(2+)-gated K(+) channels (K(Ca)) with charybdotoxin and apamin did not enhance HPV in -/- mice relative to wild-type (+/+) controls. In contrast, the voltage-gated K(+) channel (K(V)) antagonist 4-aminopyridine increased the response of -/- mice beyond that of +/+ mice. This suggested that increased K(V) channel expression could contribute to blunted HPV in PKC-epsilon -/- mice. Therefore, expression of the O(2)-sensitive K(V) channel subunit Kv3.1b (100-kDa glycosylated form and 70-kDa core protein) was compared in whole lung and pulmonary artery smooth muscle cell (PASMC) lysates from +/+ and -/- mice. A subtle increase in Kv3.1b was detected in -/- vs. +/+ whole lung lysates. A much greater rise in Kv3.1b expression was found in -/- vs. +/+ PASMC. Thus deletion of PKC-epsilon blunts murine HPV. The decreased response could not be attributed to a general loss in vasoreactivity or derangements in NOS or K(Ca) channel activity. Instead, the absence of PKC-epsilon allows increased expression of K(V) channels (like Kv3.1b) to occur in PASMC, which likely contributes to decreased HPV.     

Inducible expression of catalytically active type 1 serine/threonine protein phosphatase in a human carcinoma cell line

Background  

One of the major cellular serine/threonine protein phosphatases is protein phosphatase type 1 (PP1). Studies employing many eukaryotic systems all point to a crucial role for PP1 activity in controlling cell cycle progression. One physiological substrate for PP1 appears to be the product of the retinoblastoma susceptibility gene (pRB), a demonstrated tumor suppressor. The growth suppressive activity of pRB is regulated by its phosphorylation state. Of critical importance is the question of the in vivo effect of PP1 activity on pRB and growth regulation. As a first step towards addressing this question, we developed an inducible PP1 expression system to investigate the regulation of PP1 activity.     

DNA rearrangement in orthologous orp regions of the maize, rice and sorghum genomes

The homeologous Orp1 and Orp2 regions of maize and the orthologous regions in sorghum and rice were compared by generating sequence data for >486 kb of genomic DNA. At least three genic rearrangements differentiate the maize Orp1 and Orp2 segments, including an insertion of a single gene and two deletions that removed one gene each, while no genic rearrangements were detected in the maize Orp2 region relative to sorghum. Extended comparison of the orthologous Orp regions of sorghum and japonica rice uncovered numerous genic rearrangements and the presence of a transposon-rich region in rice. Only 11 of 27 genes (40%) are arranged in the same order and orientation between sorghum and rice. Of the 8 genes that are uniquely present in the sorghum region, 4 were found to have single-copy homologs in both rice and Arabidopsis, but none of these genes are located near each other, indicating frequent gene movement. Further comparison of the Orp segments from two rice subspecies, japonica and indica, revealed that the transposon-rich region is both an ancient and current hotspot for retrotransposon accumulation and genic rearrangement. We also identify unequal gene conversion as a mechanism for maize retrotransposon rearrangement.     

The SPRY domain-containing SOCS box protein 1 (SSB-1) interacts with MET and enhances the hepatocyte growth factor-induced Erk-Elk-1-serum response element pathway

The suppressor of cytokine signaling (SOCS) protein family includes a SPRY (repeats in splA and RyR) domain-containing SOCS box protein (SSB) subfamily, which consists of four members, SSB-1, SSB-2, SSB-3, and SSB-4. These proteins contain a central SPRY domain and a C-terminal SOCS box. Although some of the SOCS protein subfamilies function as adaptors for a large family of ubiquitin-protein isopeptide ligases to regulate certain signaling pathways, the function of the SSB subfamily remains to be determined. In our previous studies, we have found that two SPRY domain-containing proteins, RanBP9 and RanBP10, interact with MET through the SPRY domain. In the present study, we explored the function of SSB proteins in the regulation of the hepatocyte growth factor (HGF)-MET signaling. Our results showed that all four SSB proteins also interacted with the MET. The MET interaction with SSB-1 was further investigated. We demonstrated that SSB-1 bound to MET tyrosine kinase domain through its SPRY domain. MET interacted with SSB-1 in both the absence and the presence of HGF, but HGF treatment resulted in the recruitment of more SSB-1 by MET. We showed that overexpression of SSB-1 but not other SSB proteins enhanced the HGF-induced serum response element (SRE)-luciferase activity. Overexpression of SSB-1 exhibited no effect on the basal level or epidermal growth factor-induced SRE-luciferase activity. SSB-1 also enhanced HGF-induced Erk phosphorylation. Suppression of SSB-1 by the RNA interference method down-regulated HGF-induced SRE-luciferase activity and decreased Elk-1 activation. These results suggest that SSB-1 may play an important role in enhancing the HGF-induced Erk-Elk-1-SRE pathway. Furthermore, we demonstrated that in response to HGF stimulation, the SSB-1 protein became phosphorylated at tyrosine residue 31. The phosphorylated SSB-1 protein bound to p120Ras-GTPase-activating protein (GAP) but did not promote the degradation of p120RasGAP, indicating that enhanced HGF-MET signaling by overexpression of SSB-1 was not dependent on p120RasGAP degradation.     

Preservation of base-line hemodynamic function and loss of inducible cardioprotection in adult mice lacking protein kinase C epsilon

Signaling pathways involving protein kinase C isozymes are modulators of cardiovascular development and response to injury. Protein kinase C epsilon activation in cardiac myocytes reduces necrosis caused by coronary artery disease. However, it is unclear whether protein kinase C epsilon function is required for normal cardiac development or inducible protection against oxidative stress. Protein kinase C delta activation is also observed during cardiac preconditioning. However, its role as a promoter or inhibitor of injury is controversial. We examined hearts from protein kinase C epsilon knock-out mice under physiological conditions and during acute ischemia reperfusion. Null-mutant and wild-type mice displayed equivalent base-line morphology and hemodynamic function. Targeted disruption of the protein kinase C epsilon gene blocked cardioprotection caused by ischemic preconditioning and alpha(1)-adrenergic receptor stimulation. Protein kinase C delta activation increased in protein kinase C epsilon knock-out myocytes without altering resistance to injury. These observations support protein kinase C epsilon activation as an essential component of cardioprotective signaling. Our results favor protein kinase C delta activation as a mediator of normal growth. This study advances the understanding of cellular mechanisms responsible for preservation of myocardial integrity as potential targets for prevention and treatment of ischemic heart disease.