Anne Elisabeth Christensen

A novel Epac-specific cAMP analogue demonstrates independent regulation of Rap1 and ERK

cAMP is involved in a wide variety of cellular processes that were thought to be mediated by protein kinase A (PKA). However, cAMP also directly regulates Epac1 and Epac2, guanine nucleotide-exchange factors (GEFs) for the small GTPases Rap1 and Rap2 (refs 2,3). Unfortunately, there is an absence of tools to discriminate between PKA- and Epac-mediated effects. Therefore, through rational drug design we have developed a novel cAMP analogue, 8-(4-chloro-phenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphate (8CPT-2Me-cAMP), which activates Epac, but not PKA, both in vitro and in vivo. Using this analogue, we tested the widespread model that Rap1 mediates cAMP-induced regulation of the extracellular signal-regulated kinase (ERK). However, both in cell lines in which cAMP inhibits growth-factor-induced ERK activation and in which cAMP activates ERK, 8CPT-2Me-cAMP did not affect ERK activity. Moreover, in cell lines in which cAMP activates ERK, inhibition of PKA and Ras, but not Rap1, abolished cAMP-mediated ERK activation. We conclude that cAMP-induced regulation of ERK and activation of Rap1 are independent processes.

Cyclic nucleotide analogs as probes of signaling pathways

To the editor: Cyclic AMP (cAMP) and cyclic GMP (cGMP) are critical second messengers that regulate multiple targets including different cAMPor cGMP-dependent protein kinases (PKAs, PKGs)1,2, exchange proteins directly activated by cAMP (Epacs)3, phosphodiesterases (PDEs)4 and cyclic nucleotide-gated ion channels. Cyclic nucleotide analogs are widely used to study specificity of cellular signaling mediated by these target proteins. However, the selectivities and stabilities of these analogs need to be fully understood to properly interpret results and rigorously assess the mechanisms by which these analogs work in the cell. To better understand the selectivity and cross-reactivity of these analogs, we measured the activation or inhibitory activity of 13 commonly used cyclic nucleotide analogs with isozymes of PKA, PKG and Epac (Table 1), and with 8 different PDEs (Table 2 and Supplementary Tables 1 and 2 online). To measure their stability against hydrolysis, we used isothermal microcalorimetry5, a method that allowed us to evaluate whether or not an analog can function as a substrate or inhibitor for PDEs. We found that indeed some of these analogs were hydrolyzed by multiple PDEs, and other analogs were competitive inhibitors of PDEs. Here we provide half-maximal inhibition constant (Ki) data for all of the non-hydrolyzable analogs, and MichaelisMenten constant (Km) and maximum velocity (Vmax) values for all of the hydrolyzable analogs. Each of these values as well as the analog’s mode of inhibition can be determined in a single experiment (Table 2, Supplementary Methods and Supplementary Figures 1–5 online). The data strongly implied that several of these analogs might, in addition to their primary effects, also cause elevation of cAMP or cGMP indirectly by inhibiting PDEs in the cell. Such an effect could cloud interpretation of the use of these analogs. Similarly, analogs that are PDE substrates also might have their duration of action substantially reduced. To illustrate this point we showed that Sp-8-pCPT-2′O-Me-cAMPS, a highly specific, non-hydrolyzable Epac activator in vitro, can under certain conditions enhance cGMP-PKG and cAMPPKA signaling pathways in intact platelets (Supplementary Fig. 1). Specifically, we observed enhanced phosphorylation of vasodialatorstimulated phosphoprotein (VASP) at both PKA and PKG phosphorylation sites after the addition of Sp-8-pCPT-2′-O-Me-cAMPS. These data indicate that this ‘selective Epac activator’ is able to indirectly activate the cAMP-PKA and cGMP-PKG signaling pathways presumably through inhibition of platelet PDE5 and/or PDE3 (Supplementary Methods and Supplementary Discussion online). We also list in vitro selectivity data for all of the presently available commonly used cyclic nucleotide analogs for different forms of PKA, PKG and Epac I (Table 1). Data for several of these analogs have not

Epac1 and cAMP-dependent Protein Kinase Holoenzyme Have Similar cAMP Affinity, but Their cAMP Domains Have Distinct Structural Features and Cyclic Nucleotide Recognition

The cAMP-dependent protein kinase (PKA I and II) and the cAMP-stimulated GDP exchange factors (Epac1 and -2) are major cAMP effectors. The cAMP affinity of the PKA holoenzyme has not been determined previously. We found that cAMP bound to PKA I with a Kd value (2.9 μm) similar to that of Epac1. In contrast, the free regulatory subunit of PKA type I (RI) had Kd values in the low nanomolar range. The cAMP sites of RI therefore appear engineered to respond to physiological cAMP concentrations only when in the holoenzyme form, whereas Epac can respond in its free form. Epac is phylogenetically younger than PKA, and its functional cAMP site has presumably evolved from site B of PKA. A striking feature is the replacement of a conserved Glu in PKA by Gln (Epac1) or Lys (Epac2). We found that such a switch (E326Q) in site B of human RIα led to a 280-fold decreased cAMP affinity. A similar single switch early in Epac evolution could therefore have decreased the high cAMP affinity of the free regulatory subunit sufficiently to allow Epac to respond to physiologically relevant cAMP levels. Molecular dynamics simulations and cAMP analog mapping indicated that the E326Q switch led to flipping of Tyr-373, which normally stacks with the adenine ring of cAMP. Combined molecular dynamics simulation, GRID analysis, and cAMP analog mapping of wild-type and mutated BI and Epac1 revealed additional differences, independent of the Glu/Gln switch, between the binding sites, regarding space (roominess), hydrophobicity/polarity, and side chain flexibility. This helped explain the specificity of current cAMP analogs and, more importantly, lays a foundation for the generation of even more discriminative analogs.

cAMP protects neutrophils against TNF-α-induced apoptosis by activation of cAMP-dependent protein kinase, independently of exchange protein directly activated by cAMP (Epac)

It is unclear by which receptor cyclic adenosine monophosphate (cAMP) acts to promote neutrophil survival. We found that 8‐(4‐chlorophenylthio)‐2′‐O‐methyl‐cAMP, a specific activator of the recently discovered cAMP receptor, cAMP‐regulated guanosine 5′‐triphosphate exchange protein directly activated by cAMP, failed to protect human neutrophils from cell death. In contrast, specific activators of cAMP‐dependent protein kinase type I (cA‐PKI) could protect against death receptor [tumor necrosis factor receptor 1 (TNFR‐1), Fas]‐mediated apoptosis as well as cycloheximide‐accelerated “spontaneous” apoptosis. A novel “caged” cA‐PK‐activating analog, 8‐bromo (8‐Br)‐acetoxymethyl‐cAMP, was more than 20‐fold more potent than 8‐Br‐cAMP to protect neutrophils chalenged with TNF‐α against apoptosis. This analog acted more rapidly than forskolin (which increases the endogenous cAMP production) and allowed us to demonstrate that cA‐PK must be activated during the first 10 min after TNF‐α challenge to protect against apoptosis. The protective effect was mediated solely through cA‐PK activation, as it was abolished by the cA‐PKI‐directed inhibitor Rp‐8‐Br‐cAMPS and the general cA‐PK inhibitor H‐89. Neutrophils not stimulated by cAMP‐elevating agents showed increased apoptosis when exposed to the cA‐PK inhibitors Rp‐8‐Br‐cAMPS and H‐89, suggesting that even moderate activation of cA‐PK is sufficient to enhance neutrophil longevity and thereby contribute to neutrophil accumulation in chronic inflammation.

Substrate Enhances the Sensitivity of Type I Protein Kinase A to cAMP

The functional significance of the presence of two major (types I and II) isoforms of the cAMP-dependent protein kinase (PKA) is still enigmatic. The present study showed that peptide substrate enhanced the activation of PKA type I at low, physiologically relevant concentrations of cAMP through competitive displacement of the regulatory RI subunit. The effect was similar whether the substrate was a short peptide or the physiological 60-kDa protein tyrosine hydroxylase. In contrast, substrate failed to affect the cAMP-sensitivity of PKA type II. Size exclusion chromatography confirmed that substrate acted to physically enhance the dissociation of the RIα and Cα subunits of PKA type I, but not the RIIα and Cα subunits of PKA type II. Substrate availability can therefore fine-tune the activation of PKA type I by cAMP, but not PKA type II. The cAMP-dissociated RII and C subunits of PKA type II reassociated much faster than the PKA type I subunits in the presence of substrate peptide. This suggests that only PKA type II is able to rapidly reverse its activation after a burst of cAMP when exposed to high substrate concentration. We propose this as a possible reason why PKA type II is preferentially found in complexes with substrates undergoing rapid phosphorylation cycles.

A novel ADAMTSL4 mutation in autosomal recessive ectopia lentis et pupillae.

PURPOSE To examine the ocular malformations and identify the molecular genetic basis for autosomal recessive ectopia lentis et pupillae in five Norwegian families. METHODS Ten affected persons and 11 first-degree relatives of five Norwegian families underwent ophthalmic and general medical examination. Molecular genetic studies included homozygosity mapping with SNP markers, DNA sequencing, and RT-PCR analysis. RESULTS Ocular signs in affected persons were increased median corneal thickness and astigmatism, angle malformation with prominent iris processes, displacement of the pupil and lens, lens coloboma, spherophakia, loss of zonular threads, early cataract development, glaucoma, and retinal detachment. No cardiac or metabolic abnormalities known to be associated with ectopia lentis were detected. Affected persons shared a 0.67 cM region of homozygosity on chromosome 1. DNA sequencing revealed a novel mutation in ADAMTSL4, c.767_786del20. This deletion of 20 base pairs (bp) results in a frameshift and an introduction of a stop codon 113 bp downstream, predicting a C-terminal truncation of the ADAMTSL4 protein (p.Gln256ProfsX38). Expression of truncated ADAMTSL4 mRNA was confirmed by RT-PCR analysis. Three of 190 local blood donors were carriers of this mutation. CONCLUSIONS Ectopia lentis et pupillae is associated with a number of malformations primarily in the anterior segment of the eye. The causative mutation, which is the first to be described in ectopia lentis et pupillae, disrupts the same gene function previously shown to cause isolated ectopia lentis. The mutation is ancient and may, therefore, be spread to a much larger population than the investigated one.

Brittle cornea syndrome associated with a missense mutation in the zinc-finger 469 gene.

PURPOSE To investigate the diverse clinical manifestations, identify the causative mutation and explain the association with red hair in a family with brittle cornea syndrome (BCS). METHODS Eight family members in three generations underwent ophthalmic, dental, and general medical examinations, including radiologic examination of the spine. Bone mineral density (BMD) and serum levels of vitamin D, parathyroid hormone, and biochemical markers for bone turnover were measured. Skin biopsies were examined by light and transmission electron microscopy. Molecular genetic studies included homozygosity mapping with SNP markers, DNA sequencing, and MC1R genotyping. RESULTS At 42 and 48 years of age, respectively, both affected individuals were blind due to retinal detachment and secondary glaucoma. They had extremely thin and bulging corneas, velvety skin, chestnut colored hair, scoliosis, reduced BMD, dental anomalies, hearing loss, and minor cardiac defects. The morphologies of the skin biopsies were normal except that in some areas slightly thinner collagen fibrils were seen in one of the affected individuals. Molecular genetic analysis revealed a novel missense mutation of ZNF469, c.10016G>A, that was predicted to affect the fourth of the five zinc finger domains of ZNF469 by changing the first cysteine to a tyrosine (p.Cys3339Tyr). Both affected individuals were homozygous for the common red hair variant R151C at the MC1R locus. CONCLUSIONS BCS is a disorder that affects a variety of connective tissues. Reduced BMD and atypical dental crown morphology have not been reported previously. The results confirm that BCS is associated with mutations in ZNF469. The association with red hair in some individuals with BCS is likely to occur by chance.

CHAPTER 212 – Cyclic Nucleotide Analogs as Tools to Investigate Cyclic Nucleotide Signaling

Early after the discovery of the two naturally occurring cyclic nucleotide monophosphates (cNMPs) cAMP and cGMP, the lipophilic analog N 6 , 2′-O-dibutyryl cAMP was synthesized and used to elicit cAMP responses in intact cells. Several hundred cNMP analogs have since been synthesized. This chapter illustrates the guidelines and examples of the use of cNMP analogs and describes the chemistry and some properties of commonly used cNMP analogs. Currently available cNMP analogs have proven to be very useful tools in cell biology. There is nevertheless room for considerable progress regarding both the synthesis of more specific and potent analogs with improved pharmacokinetic properties and mapping of the cNMP receptors. An interesting novel approach is to synthesize polymer-linked cNMPs to simultaneously occupy two binding sites in the same receptor complex.

Cyclic Nucleotide Analogs as Tools to Investigate Cyclic Nucleotide Signaling

Publisher Summary The two naturally occurring cyclic nucleotide monophosphates (cNMPs) cAMP and cGMP serve as paradigms for second-messenger cell signaling. The effects of cAMP in vertebrates appear to be mediated mainly through activation of cAMP-dependent protein kinase isozymes (cA-PKI and cA-PKII), but also through activation of the small GTPase exchange factors (Epac1 and Epac 2) and direct binding to ion channels. The parent cAMP and cGMP molecules are virtually unable to penetrate cell membranes by diffusion, and are readily broken down by cyclic nucleotide phosphodiesterases. The cyclic phosphate ring is essential for the cNMP affinity to its binding sites, cAMP having seven orders of magnitude higher affinity than AMP for cA-PK. The use of activatory cNMP analogs can provide independent evidence that a cyclic nucleotide elevating agent acts via cAMP or cGMP. The cellular accumulation of cNMP analogs depends on the balance between inward and outward transport through membrane pores, on the rate of diffusion through the surface membrane, and on the analog stability within the cell. In general, moderately lipophilic analogs have better bioavailability than hydrophilic analogs, presumably due to better transmembrane diffusion. cNMP analogs are generally much poorer substrates than the parent cAMP or cGMP for cyclic nucleotide phopshodiesterases and their most serious side effects are due to their metabolic products.