Juniper Pennypacker
University of California, San Diego
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Featured researches published by Juniper Pennypacker.
Journal of Biological Chemistry | 2010
Hemal H. Patel; Lora L. Hamuro; Byeong Jo Chun; Yoshitaka Kawaraguchi; Alexander Quick; Brian Rebolledo; Juniper Pennypacker; Jackie Thurston; Natalia Rodriguez-Pinto; Christopher Self; Gary E. Olson; Paul A. Insel; Wayne R. Giles; Susan S. Taylor; David Roth
Localization of protein kinase A (PKA) via A-kinase-anchoring proteins (AKAPs) is important for cAMP responsiveness in many cellular systems, and evidence suggests that AKAPs play an important role in cardiac signaling. To test the importance of AKAP-mediated targeting of PKA on cardiac function, we designed a cell-permeable peptide, which we termed trans-activator of transcription (TAT)-AKAD for TAT-conjugated A-kinase-anchoring disruptor, using the PKA binding region of AKAP10 and tested the effects of this peptide in isolated cardiac myocytes and in Langendorff-perfused mouse hearts. We initially validated TAT-AKAD as a PKA localization inhibitor in cardiac myocytes by the use of confocal microscopy and cellular fractionation to show that treatment with the peptide disrupts type I and type II PKA regulatory subunits. Knockdown of PKA activity was demonstrated by decrease in phosphorylation of phospholamban and troponin I after β-adrenergic stimulation in isolated myocytes. Treatment with TAT-AKAD reduced myocyte shortening and rates of contraction and relaxation. Injection of TAT-AKAD (1 μm), but not scrambled control peptide, into the coronary circulation of isolated perfused hearts rapidly (<1 min) and reversibly decreased heart rate and peak left ventricular developed pressure. TAT-AKAD also had a pronounced effect on developed pressure (−dP/dt), consistent with a delayed relaxation of the heart. The effects of TAT-AKAD on heart rate and contractility persisted in hearts pretreated with isoproterenol. Disruption of PKA localization with TAT-AKAD thus had negative effects on chronotropy, inotropy, and lusitropy, thereby indicating a key role for AKAP-targeted PKA in control of heart rate and contractile function.
Journal of Biological Chemistry | 2007
Kenneth M. Humphries; Juniper Pennypacker; Susan S. Taylor
Many components of cellular signaling pathways are sensitive to regulation by oxidation and reduction. Previously, we described the inactivation of cAMP-dependent protein kinase (PKA) by direct oxidation of a reactive cysteine in the activation loop of the kinase. In the present study, we demonstrate that in HeLa cells PKA activity follows a biphasic response to thiol oxidation. Under mild oxidizing conditions, or short exposure to oxidants, forskolin-stimulated PKA activity is enhanced. This enhancement was blocked by sulfhydryl reducing agents, demonstrating a reversible mode of activation. In contrast, forskolin-stimulated PKA activity is inhibited by more severe oxidizing conditions. Mild oxidation enhanced PKA activity stimulated by forskolin, isoproterenol, or the cell-permeable analog, 8-bromo-cAMP. When cells were lysed in the presence of serine/threonine phosphatase inhibitor, NaF, the PKA-enhancing effect of oxidation was blunted. These results suggest oxidation of a PKA-counteracting phosphatase may be inhibited, thus enhancing the apparent kinase activity. Using an in vivo PKA activity reporter, we demonstrated that mild oxidation does indeed prolong the PKA signal induced by isoproterenol by inhibiting counteracting phosphatase activity. The results of this study demonstrate in live cells a unique synergistic mechanism whereby the PKA signaling pathway is enhanced in an apparent biphasic manner.
Journal of Biological Chemistry | 2009
Jie Yang; Eileen J. Kennedy; Jian Wu; Michael S. Deal; Juniper Pennypacker; Gourisankar Ghosh; Susan S. Taylor
Protein kinase A holoenzyme is comprised of two catalytic (C) and two regulatory (R) subunits which keep the enzyme in an inhibited state before activation by cyclic-AMP. The C-subunit folds into a conserved bi-lobal core flanked by N- and C-terminal tails. We report here characterization of a C-tail loss-of-function mutant, CF327A, and a related suppressor mutant, CF327A/K285P. Phe-327 is the only residue outside the kinase core that binds to the adenine ring of ATP, whereas Lys-285 is ∼45 Å away and lies in an AGC kinase-specific insert. The two mutations were previously identified from a yeast genetic screen, where the F327A mutation was unable to complement cell growth but mutation of K285P in the same allele rescued cell viability. We show that CF327A exhibits significant reduction in catalytic efficiency, which likely explains the observed loss-of-function phenotype. Interestingly, the additional K285P mutation does not restore kinase activity but reduces the inhibitory interaction of the double mutant with RII subunits. The additional K285P mutation, thus, helps to keep a low but uninhibited PKA activity that is sufficient for cell viability. The crystal structure of CF327A/K285P further reveals that recruitment of Phe-327 to the ATP binding pocket not only contributes to the hydrophobic pocket, as previously thought, but also recruits its flanking C-tail region to the kinase core, thereby concertedly positioning the glycine-rich loop and ATP for phosphoryl transfer. The study exemplifies two different ways for regulating cAMP-dependent protein kinase activity through non-conserved residues and sheds light on the structural and functional diversity of the kinase family.
PLOS ONE | 2011
Charles C. King; Mira Sastri; Philip Chang; Juniper Pennypacker; Susan S. Taylor
The mechanism of PKAc-dependent NF-κB activation and subsequent translocation into the nucleus is not well defined. Previously, we showed that A kinase interacting protein 1 (AKIP1) was important for binding and retaining PKAc in the nucleus. Since then, other groups have demonstrated that AKIP1 binds the p65 subunit of NF-κB and regulates its transcriptional activity through the phosphorylation at Ser 276 by PKAc. However, little is known about the formation and activation of the PKAc/AKIP1/p65 complex and the rate at which it enters the nucleus. Initially, we found that the AKIP1 isoform (AKIP 1A) simultaneously binds PKAc and p65 in resting and serum starved cells. Using peptide arrays, we refined the region of AKIP 1A binding on PKAc and mapped the non-overlapping regions on AKIP 1A where PKAc and p65 bind. A peptide to the amino-terminus of PKAc (CAT 1-29) was generated to specifically disrupt the interaction between AKIP 1A and PKAc to study nuclear import of the complex. The rate of p65 nuclear translocation was monitored in the presence or absence of overexpressed AKIP 1A and/or (CAT 1-29). Enhanced nuclear translocation of p65 was observed in the presence of overexpressed AKIP1 and/or CAT 1-29 in cells stimulated with TNFα, and this correlated with decreased phosphorylation of serine 276. To determine whether PKAc phosphorylation of p65 in the cytosol regulated nuclear translocation, serine 276 was mutated to alanine or aspartic acid. Accelerated nuclear accumulation of p65 was observed in the alanine mutant, while the aspartic acid mutation displayed slowed nuclear translocation kinetics. In addition, enhanced nuclear translocation of p65 was observed when PKAc was knocked-down by siRNA. Taken together, these results suggest that AKIP 1A acts to scaffold PKAc to NF-κB in the cytosol by protecting the phosphorylation site and thereby regulating the rate of nuclear translocation of p65.
Protein Science | 2006
Dominico Vigil; Jung-Hsin Lin; Christoph A. Sotriffer; Juniper Pennypacker; J. Andrew McCammon; Susan S. Taylor
Cyclic AMP activates protein kinase A by binding to an inhibitory regulatory (R) subunit and releasing inhibition of the catalytic (C) subunit. Even though crystal structures of regulatory and catalytic subunits have been solved, the precise molecular mechanism by which cyclic AMP activates the kinase remains unknown. The dynamic properties of the cAMP binding domain in the absence of cAMP or C‐subunit are also unknown. Here we report molecular‐dynamics simulations and mutational studies of the RIα R‐subunit that identify the C‐helix as a highly dynamic switch which relays cAMP binding to the helical C‐subunit binding regions. Furthermore, we identify an important salt bridge which links cAMP binding directly to the C‐helix that is necessary for normal activation. Additional mutations show that a hydrophobic “hinge” region is not as critical for the cross‐talk in PKA as it is in the homologous EPAC protein, illustrating how cAMP can control diverse functions using the evolutionarily conserved cAMP‐binding domains.
Structure | 2011
Angela J. Boettcher; Jian Wu; Choel Kim; Jie Yang; Jessica G.H. Bruystens; Nikki Cheung; Juniper Pennypacker; Donald A. Blumenthal; Alexandr P. Kornev; Susan S. Taylor
Biochemistry | 2003
Lora L. Burns; Jaume M. Canaves; Juniper Pennypacker; Donald K. Blumenthal; Susan S. Taylor
Structure | 2011
Angela J. Boettcher; Jian Wu; Choel Kim; Jie Yang; Jessica G.H. Bruystens; Nikki Cheung; Juniper Pennypacker; D.A Blumenthal; Alexandr P. Kornev; Susan S. Taylor
Archive | 2010
Hemal H. Patel; Lora L. Hamuro; Byeong Jo Chun; Yoshitaka Kawaraguchi; Alexander Quick; Brian Rebolledo; Juniper Pennypacker; Jackie Thurston; Natalia Rodriguez-Pinto; Christopher Self; Gary E. Olson; Paul A. Insel; Wayne R. Giles; Susan S. Taylor; David Roth; FromtheDepartmentsof ‡ Anesthesiology
The FASEB Journal | 2008
Jie Yang; Eileen Kenndy; Jian Wu; Michael S. Deal; Simon H. J. Brown; Juniper Pennypacker; Gourisankar Ghosh