Regulation of actin dynamics by protein kinase R control of gelsolin enforces basal innate immune defense.

Primary resistance to pathogens is reliant on both basal and inducible immune defenses. To date, research has focused upon inducible innate immune responses. In contrast to resistance via cytokine induction, basal defense mechanisms are less evident. Here we showed that the antiviral protein kinase R (PKR) inhibited the key actin-modifying protein gelsolin to regulate actin dynamics and control cytoskeletal cellular functions under homeostatic conditions. Through this mechanism, PKR controlled fundamental innate immune, actin-dependent processes that included membrane ruffling and particle engulfment. Accordingly, PKR counteracted viral entry into the cell. These findings identify a layer of host resistance, showing that the regulation of actin-modifying proteins during the innate immune response bolsters first-line defense against intracellular pathogens and has a sustained effect on virus production. Moreover, these data provide proof of principle for a concept in which the cell cytoskeleton could be targeted to elicit broad antiviral protection.

The extreme C-terminal region of phospholipase Cbeta1 determines subcellular localization and function; the "b" splice variant mediates alpha1-adrenergic receptor responses in cardiomyocytes.

Phospholipase Cbeta1 (PLCbeta1) exists as two splice variants, PLCbeta1a (150 kDa) and PLCbeta1b (140 kDa), which differ only in their C-terminal sequences of 64 and 31 amino acids, respectively. The 3 C-terminal amino acid residues of PLCbeta1a comprise a PDZ-interacting domain, whereas the PLCbeta1b sequence has no PDZ-interacting domain but contains unique proline-rich domain 5 residues from the C terminus. PLCbeta1a is localized in the cytoplasm, whereas PLCbeta1b targets to the sarcolemma and is enriched in caveolae. Deletion of 3 amino acids from the C terminus of PLCbeta1b did not alter its sarcolemmal localization, but deletion of the entire unique 31 amino acid sequence caused cytosolic localization. A myristoylated 10 amino acid peptide from the C terminus of PLCbeta1b selectively dissociated N-terminally GFP-tagged PLCbeta1b from the sarcolemma and inhibited PLC responses to alpha(1)-adrenergic agonists, with a half maximal effective concentration of 12 +/- 1.6 microM (mean+/-SE, n=3). A similar peptide from PLCbeta1a was without effect at concentrations below 100 microM. Thus, the extreme C-terminal sequences of the PLCbeta1 splice variants determine localization and, thus, function. In cardiomyocytes, responses initiated by alpha(1)-adrenergic receptor activation involve only PLCbeta1b, and the selective targeting of this splice variant to the sarcolemma provides a potential therapeutic target to reduce hypertrophy, apoptosis, and arrhythmias.

Ins(1,4,5)P(3) regulates phospholipase Cbeta1 expression in cardiomyocytes.

The functional significance of the Ca2+-releasing second messenger inositol(1,4,5)trisphosphate (Ins(1,4,5)P(3), IP(3)) in the heart has been controversial. Ins(1,4,5)P(3) is generated from the precursor lipid phosphatidylinositol(4,5)bisphosphate (PIP(2)) along with sn-1,2-diacylglycerol, and both of these are important cardiac effectors. Therefore, to evaluate the functional importance of Ins(1,4,5)P(3) in cardiomyocytes (NRVM), we overexpressed IP(3) 5-phosphatase to increase degradation. Overexpression of IP(3) 5-phosphatase reduced Ins(1,4,5)P(3) responses to alpha(1)-adrenergic receptor agonists acutely, but with longer stimulation, caused an overall increase in phospholipase C (PLC) activity, associated with a selective increase in expression of PLCbeta1, that served to normalise Ins(1,4,5)P(3) content. Similar increases in PLC activity and PLCbeta1 expression were observed when Ins(1,4,5)P(3) was sequestered onto the PH domain of PLCdelta1, a high affinity selective Ins(1,4,5)P(3)-binding motif. These findings suggested that the available level of Ins(1,4,5)P(3) selectively regulates the expression of PLCbeta1. Cardiac responses to Ins(1,4,5)P(3) are mediated by type 2 IP(3)-receptors. Hearts from IP(3)-receptor (type 2) knock-out mice showed heightened PLCbeta1 expression. We conclude that Ins(1,4,5)P(3) and IP(3)-receptor (type 2) regulate PLCbeta1 and thereby maintain levels of Ins(1,4,5)P(3). This implies some functional significance for Ins(1,4,5)P(3) in the heart.

Alpha1-adrenergic receptor signaling is localized to caveolae in neonatal rat cardiomyocytes.

In neonatal rat cardiomyocytes, phosphatidylinositol(4,5)bisphosphate (PIP2) is a precursor of second messengers, a stabilizer of ion channels and exchangers, an anchor point for the cytoskeleton and, in addition, can serve as a signaling molecule in its own right. We examined the possibility that sarcolemmal PIP2 exists in different pools and that only one of these provides the substrate for alpha1-adrenergic receptor activated phospholipase C (PLC). Membranes were separated on the basis of buoyant density, and the light lipid raft fractions were further separated into caveolae and non-caveolar rafts using immunoprecipitation. PIP2 was principally located in the light lipid raft fractions and was equally distributed between caveolae and non-caveolar membranes. Heavier membrane fractions also contained some PIP2. Addition of the alpha1-adrenergic receptor agonist phenylephrine (50 microM) caused reductions in PIP2, but only in caveolae. PIP2 in other fractions was unaffected. In agreement with this, PLCbeta1 and, to a lesser extent, Galphaq were concentrated in this fraction. PLCbeta3 was primarily observed in heavier membranes. We conclude that PIP2 in cardiomyocyte sarcolemma is compartmentalized and that alpha1-adrenergic receptor signaling is localized to caveolae.