PDE4B and PDE4D provide >90% of PDE4 cAMP phosphodiesterase activity in human embryonic kidney (HEK293B2) cells. Their selective small interference RNA (siRNA)-mediated knockdown potentiates isoprenaline-stimulated protein kinase A (PKA) activation. Whereas endogenous PDE4D co-immunoprecipitates with ␤arrestin, endogenous PDE4B does not, even upon PDE4D knockdown. Ectopic overexpression of PDE4B2 confers co-immunoprecipitation with ␤arrestin. Knockdown of PDE4D, but not PDE4B, amplifies isoprenaline-stimulated phosphorylation of the ␤ 2 -adrenergic receptor (␤ 2 -AR) by PKA and activation of extracellular signal-regulated kinase (ERK) through G i . Isoform-selective knockdown identifies PDE4D5 as the functionally important species regulating isoprenaline stimulation of both these processes. Ht31-mediated disruption of the tethering of PKA to AKAP scaffold proteins attenuates isoprenaline activation of ERK, even upon PDE4D knockdown. Selective siRNA-mediated knockdown identifies AKAP79, which is constitutively associated with the ␤ 2 -AR, rather than isoprenaline-recruited gravin, as being the functionally relevant AKAP in this process. Isoprenaline-stimulated membrane recruitment of PDE4D is ablated upon ␤arrestin knockdown. A mutation that compromises interactions with ␤arrestin prevents catalytically inactive PDE4D5 from performing a dominant negative role in potentiating isoprenaline-stimulated ERK activation. ␤arrestin-recruited PDE4D5 desensitizes isoprenaline-stimulated PKA phosphorylation of the ␤ 2 -AR and the consequential switching of its signaling to ERK. The ability to observe a cellular phenotype upon PDE4D5 knockdown demonstrates that other PDE4 isoforms, expressed at endogenous levels, are unable to afford rescue in HEK293B2 cells.It is now well appreciated that cAMP signaling is compartmentalized in cells (1-4). This notion arose originally from elegant studies done on cardiac myocytes (2) and gained considerable credence because the discovery of anchor proteins (AKAPs) for protein kinase A (PKA) 4 allowed gradients of cAMP to be sensed and acted upon accordingly (1, 4, 5). More recently, a number of cAMP sensors have been developed that have allowed gradients of cAMP to be identified and even visualized in cells (6 -9). Paramount to the generation and shaping of intracellular gradients is the action of cAMP phosphodiesterases, which provide the sole means of degrading cAMP in cells (2, 10 -14). Of these, the PDE4 family of enzymes has gained attention in view of the fact that chemical knock-out with selective inhibitors indicates that they perform an important role in regulating key processes such as inflammation and cognition (15)(16)(17)(18)(19)(20). Four genes (4A, 4B, 4C, and 4D) encode a large family of PDE4 isoforms (15,17). Little is known, however, about the functional significance of each PDE4 sub-family. Nevertheless, important insights into physiological function have come from gene knockout studies on the PDE4B and PDE4D sub-families, which have implied distinct roles for these sub-f...

The factors contributing to selective motoneuron loss in amyotrophic lateral sclerosis (ALS) remain undefined. To investigate whether calcium-binding proteins contribute to selective motoneuron vulnerability in ALS, we compared calbindin-D28K and parvalbumin immunoreactivity in motoneuron populations in human ALS, and in a ventral spinal cord hybrid cell line selectively vulnerable to the cytotoxic effects of ALS IgG. In human autopsy specimens, immunoreactive calbindin-D28k and parvalbumin were absent in motoneuron populations lost early in ALS (i.e., cortical and spinal motoneurons, lower cranial nerve motoneurons), while motoneurons damaged late or infrequently in the disease (i.e., Onuf's nucleus motoneurons, oculomotor, trochlear, and abducens nerve neurons) expressed markedly higher levels of immunoreactive calbindin-D28K and/or parvalbumin. Motoneuron-neuroblastoma VSC 4.1 hybrid cells lost immunoreactive calbindin-D28k and parvalbumin following dibutyryl-cyclic AMP-induced differentiation and were killed by IgG from ALS patients. Undifferentiated calbindin/parvalbumin-reactive VSC 4.1 cells were not killed, nor were other cell lines expressing high levels of calbindin-D28K and parvalbumin immunoreactivity (substantia nigra-neuroblastoma hybrid cells and N18TG2 neuroblastoma parent cells). These studies suggest that decreased calbindin-D28K and parvalbumin immunoreactivity may help explain the selective vulnerability of motoneurons in ALS.

The cAMP-specific phosphodiesterase PDE4D5 can interact with the signalling scaffold proteins RACK (receptors for activated C-kinase) 1 and beta-arrestin. Two-hybrid and co-immunoprecipitation analyses showed that RACK1 and beta-arrestin interact with PDE4D5 in a mutually exclusive manner. Overlay studies with PDE4D5 scanning peptide array libraries showed that RACK1 and beta-arrestin interact at overlapping sites within the unique N-terminal region of PDE4D5 and at distinct sites within the conserved PDE4 catalytic domain. Screening scanning alanine substitution peptide arrays, coupled with mutagenesis and truncation studies, allowed definition of RACK1 and beta-arrestin interaction sites. Modelled on the PDE4D catalytic domain, these form distinct well-defined surface-exposed patches on helices-15-16, for RACK1, and helix-17 for beta-arrestin. siRNA (small interfering RNA)-mediated knockdown of RACK1 in HEK-293 (human embryonic kidney) B2 cells increased beta-arrestin-scaffolded PDE4D5 approx. 5-fold, increased PDE4D5 recruited to the beta2AR (beta2-adrenergic receptor) upon isoproterenol challenge approx. 4-fold and severely attenuated (approx. 4-5 fold) both isoproterenol-stimulated PKA (protein kinase A) phosphorylation of the beta2AR and activation of ERK (extracellular-signal-regulated kinase). The ability of a catalytically inactive form of PDE4D5 to exert a dominant negative effect in amplifying isoproterenol-stimulated ERK activation was ablated by a mutation that blocked the interaction of PDE4D5 with beta-arrestin. In the present study, we show that the signalling scaffold proteins RACK1 and beta-arrestin compete to sequester distinct 'pools' of PDE4D5. In this fashion, alterations in the level of RACK1 expression may act to modulate signal transduction mediated by the beta2AR.

We identify a compartmentalized signaling system that identifies a functional role for the GTP exchange factor, exchange protein activated by cAMP (EPAC) coupled to Rap2 in the nucleus. In this system, cAMP regulates the nuclear/cytoplasmic trafficking of DNA-dependent protein kinase (DNA-PK), a critical kinase that acts to repair double-stranded breaks (DSBs) in damaged DNA and to phosphorylate the cell survival kinase, PKB/Akt. Intersecting regulatory inputs for cAMP employ EPAC to transduce positive effects, namely the Rap2-dependent nuclear exit and activation of DNA-PK, whereas protein kinase A (PKA) provides the negative input by antagonizing these actions. We identify this as a compartmentalized regulatory system where modulation of cAMP input into the stimulatory, EPAC and inhibitory, PKA intersecting arms is provided by spatially discrete, cAMP degradation systems. The distribution of DNA-PK between nuclear and cytoplasmic compartments can thus potentially be influenced by relative inputs of cAMP signaling through the EPAC and PKA pathways. Through this signaling system EPAC activation can thereby impact on the Ser-473 phosphorylation status of PKB/Akt and the repair of etoposide-induced DSBs.phosphodiesterase ͉ PDE4 ͉ rolipram ͉ PKB ͉ Akt

Intracellular calcium is increased in vulnerable spinal motoneurons in immune-mediated as well as transgenic models of amyotrophic lateral sclerosis (ALS). To determine whether intracellular calcium levels are in¯uenced by the calciumbinding protein parvalbumin, we developed transgenic mice overexpressing parvalbumin in spinal motoneurons. ALS immunoglobulins increased intracellular calcium and spontaneous transmitter release at motoneuron terminals in control animals, but not in parvalbumin overexpressing transgenic mice. Parvalbumin transgenic mice interbred with mutant SOD1 (mSOD1) transgenic mice, an animal model of familial ALS, had signi®cantly reduced motoneuron loss, and had delayed disease onset (17%) and prolonged survival (11%) when compared with mice with only the mSOD1 transgene. These results af®rm the importance of the calcium binding protein parvalbumin in altering calcium homeostasis in motoneurons. The increased motoneuron parvalbumin can signi®-cantly attenuate the immune-mediated increases in calcium and to a lesser extent compensate for the mSOD1-mediated toxic-gain-of-function' in transgenic mice.