Subcellular targeting of signaling enzymes influences where and when various modes of intracellular communication operate. Macromolecular complexes of signal transduction and signal termination elements favor reversible control of repetitive processes. This includes adrenergic stimulation of excitation-contraction coupling in the heart. Long isoforms of A-kinase anchoring protein 18 (AKAP18γ and δ) modulate this process via regulation of calcium uptake into the sarcoplasmic reticulum through the Ca2+ATPase 2a (SERCA2a). AKAP18 proximity-proteomic screening in cardiomyocytes identifies networks for protein kinase A (PKA) and ubiquitin-specific proteinases (USPs). A 2'phosphoesterase domain on AKAP18 interfaces with the USP4 isoform at the Z bands of sarcomeres. PKA stimulates USP4 activity in the presence of the anchoring protein. AKAP18 anchored PKA phosphorylates serine 829 on USP4, a conserved residue near the active site of this deubiquitinase. Antibodies against the pSer829 motif show that adrenergic stimulation enhances phosphorylation of USP4 in mouse adult cardiomyocytes. In related studies, elevated USP4 phosphorylation at Ser829 is detected in human post myocardial infraction tissue as compared to healthy tissue. Thus, phosphorylation of sarcoplasmic USP4 may be a cardioprotective response. Pharmacological inhibition of PKA or deletion of the AKAP7/18 gene in mice decreases calcium flux through the exchanger. This suggests that loss of the anchoring protein impacts SERCA2 action. Thus, AKAP18/PKA/USP4 complexes are well positioned to influence the rate and magnitude of calcium reuptake during the cardiac cycle.

Adrenal Cushing’s syndrome is a disease of cortisol hypersecretion often caused by mutations in protein kinase A catalytic subunit (PKAc). Using a personalized medicine screening platform, we discovered a Cushing’s driver mutation, PKAc-W196G, in ~20% of patient samples analyzed. Proximity proteomics and photokinetic imaging reveal that PKAc W196G is unexpectedly distinct from other described Cushing’s variants, exhibiting retained association with type I regulatory subunits (RI) and their corresponding A kinase anchoring proteins (AKAPs). Molecular dynamics simulations predict that substitution of tryptophan-196 with glycine creates a 653–cubic angstrom cleft between the catalytic core of PKAc W196G and type II regulatory subunits (RII), but only a 395–cubic angstrom cleft with RI. Endocrine measurements show that overexpression of RIα or redistribution of PKAc W196G via AKAP recruitment counteracts stress hormone overproduction. We conclude that a W196G mutation in the kinase catalytic core skews R subunit selectivity and biases AKAP association to drive Cushing’s syndrome.

Clearance of invading pathogens is essential to preventing overwhelming inflammation and sepsis that are symptomatic of bacterial peritonitis. Macrophages participate in this innate immune response by engulfing and digesting pathogens, a process called phagocytosis. Oxidized phospholipids (OxPL) are danger-associated molecular patterns (DAMPs) generated in response to infection that can prevent the phagocytic clearance of bacteria. We investigated the mechanism underlying OxPL action in macrophages. Exposure to OxPL induced alterations in actin polymerization, resulting in spreading of peritoneal macrophages and diminished uptake of E. coli. Pharmacological and cell-based studies showed that an anchored pool of PKA mediates the effects of OxPL. Gene silencing approaches identified the A-kinase anchoring protein (AKAP) WAVE1 as an effector of OxPL action in vitro. Chimeric Wave1–/– mice survived significantly longer after infection with E. coli and OxPL treatment in vivo. Moreover, we found that endogenously generated OxPL in human peritoneal dialysis fluid from end-stage renal failure patients inhibited phagocytosis via WAVE1. Collectively, these data uncover an unanticipated role for WAVE1 as a critical modulator of the innate immune response to severe bacterial infections. Research Article Immunology

Podokinetic after-rotation (PKAR) is a phenomenon in which subjects inadvertently rotate when instructed to step in place after a period of walking on a rotating treadmill. PKAR has been shown to transfer between different forms of locomotion, but has not been tested in a non-locomotor task. We conducted two experiments to assess effects of PKAR on perception of subjective straight ahead and on quiet standing posture. Twenty-one healthy young right-handed subjects pointed to what they perceived as their subjective straight ahead with a laser pointer while they were recorded by a motion capture system both before and after a training period on the rotating treadmill. Subjects performed the pointing task while standing, sitting on a chair without a back, and a chair with a back. After the training period, subjects demonstrated a significant shift in subjective straight ahead, pointing an average of 29.1 ± 10.6° off of center. The effect was direction-specific, depending on whether subjects had trained in the clockwise or counter-clockwise direction. Postures that limited subjects’ ability to rotate the body in space resulted in reduction, but not elimination, of the effect. The effect was present in quiet standing and even in sitting postures where locomotion was not possible. The robust transfer of PKAR to non-locomotor tasks, and across locomotor forms as demonstrated previously, is in contrast to split-belt adaptations that show limited transfer. We propose that, unlike split-belt adaptations, podokinetic adaptations are mediated at supraspinal, spatial orientation areas that influences spinal-level circuits for locomotion.

www.sciencemag.org (this information is current as of February 4, 2009 ): The following resources related to this article are available online at http://www.sciencemag.org/cgi/content/full/285/5424/93 version of this article at: including high-resolution figures, can be found in the online Updated information and services, http://www.sciencemag.org/cgi/content/full/285/5424/93#otherarticles , 22 of which can be accessed for free: cites 41 articles This article 269 article(s) on the ISI Web of Science. cited by This article has been http://www.sciencemag.org/cgi/content/full/285/5424/93#otherarticles 100 articles hosted by HighWire Press; see: cited by This article has been http://www.sciencemag.org/cgi/collection/cell_biol Cell Biology : subject collections This article appears in the following http://www.sciencemag.org/about/permissions.dtl in whole or in part can be found at: this article permission to reproduce of this article or about obtaining reprints Information about obtaining

D The activity of the transcription factor hypoxia-inducible factor 1a (HIF-1a) is increased in response to reduced intracellular oxygen. Enzymes of the protein ubiquitin machinery that signal the destruction or stabilization of HIF-1a tightly control this transcriptional response. Here, we show that muscle A kinase–anchoring protein (mAKAP) organized ubiquitin E3 ligases that managed the stability of HIF1a and optimally positioned it close to its site of action inside the nucleus. Functional experiments in cardiomyocytes showed that depletion of mAKAP or disruption of its targeting to the perinuclear region altered the stability of HIF-1a and transcriptional activation of genes associated with hypoxia. Thus, we propose that compartmentalization of oxygen-sensitive signaling components may influence the fidelity and magnitude of the hypoxic response. ow

Susceptibility to mouse plasmacytomagenesis is a complex genetic trait controlled by several Pctr loci (Pctr1, Pctr2, etc). Congenic strain analysis narrowed the genetic interval surrounding the Pctr2 locus, and genes identified in the interval were sequenced from susceptible BALB/c and resistant DBA/2 mice. Frap (FKBP12 rapamycin-associated protein, mTOR, RAFT) was the only gene differing in amino acid sequence between alleles that correlated with strain sensitivity to tumor development. The in vitro kinase activity of the BALB/c FRAP allele was lower than the DBA/2 allele; phosphorylation of p53 and PHAS1/4EBP1 (properties of heat and acid stability/eukaryotic initiation factor 4E-binding protein) and autophosphorylation of FRAP were less efficient with the BALB/c allele. FRAP also suppressed transformation of NIH 3T3 cells by ras, with DBA/2 FRAP being more efficient than BALB/c FRAP. Rapamycin, a specific inhibitor of FRAP, did not inhibit growth of plasmacytoma cell lines. These studies identify Frap as a candidate tumor suppressor gene, in contrast to many reports that have focused on its prooncogenic properties. Frap may be similar to Tgfb and E2f in exerting both positive and negative growth-regulatory signals, depending on the timing, pathway, or tumor system involved. The failure of rapamycin to inhibit plasma cell tumor growth suggests that FRAP antagonists may not be appropriate for the treatment of plasma cell tumors. Pctr2 joins Pctr1 in possessing alleles that modify susceptibility to plasmacytomagenesis by encoding differences in efficiency of function (efficiency alleles), rather than all-or-none, gain-of-function, or loss-of-function alleles. By analogy, human cancer may also result from the combined effects of several inefficient alleles.

The deduced amino acid sequence of anti-fluorescein (F1) antibody 3–13 VH region (residues 1–95) was 78% homologous to the α-1→ 3-dextran binding myeloma protein J558 vh region and was in the Wu-Kabat Subgroup II or Dildrop Group I classifications. The 3–13 VH region was rearranged to a D segment with only 8 of 30 bp in common with DFL16.1 germ line D gene and less homologous to all other previousy identified D sequences. This sequence was joined to the third codon of JH4. The sequence encoding VH residues 5–91 was subcloned into pSP65 and used as a probe in Southern analyses to monitor 3–13 VH gene rearrangements in 12 other anti-F1 hybridomas differentially expressing (or not at all) the 3–13 idiotype. Three clones which inhibited the 3–13 idiotype-anti-idiotype interaction as effectively as 3–13 (3–12, 3–17 and 3–35), all had rearranged a gene which hybridized to the cloned 3–13 fragment, however, each was contained on a different size restriction fragment. Analyses of five other idiotypically related (but not identical) hybridomas indicated that four had rearranged a cross hybridizing VHgene while no such rearrangements were detected among four idiotyplcally negative cell lines. A restriction site assay indicated five clones examined had all rearranged a Vκ gene to the Jκ 1 or Jκ2 gene segment. The sequence of the antibody 3–13 VH gene and its use in hybridization studies represent the first molecular analysis of a recurrently expressed repertoire specific idiotype within an unrestricted immune response.