Michael D. Uhler

Cyclic AMP-dependent protein kinase decreases GABAA receptor current in mouse spinal neurons

GABA, the major inhibitory neurotransmitter in the mammalian brain, binds to GABAA receptors, which form chloride ion channels. The predicted structure of the GABAA receptor places a consensus phosphorylation site for cAMP-dependent protein kinase (PKA) on an intracellular domain of the channel. Phosphorylation by various protein kinases has been shown to alter the activity of certain ligand- and voltage-gated ion channels. We have examined the role of phosphorylation by the catalytic subunit of PKA in the regulation of GABAA receptor channel function using whole-cell and excised outside-out patch-clamp techniques. Inclusion of the catalytic subunit of PKA in the recording pipettes significantly reduced GABA-evoked whole-cell and single-channel chloride currents. Both heat inactivation of PKA and addition of the specific protein kinase inhibitor peptide prevented the reduction of GABA-evoked currents by PKA. Neither mean channel open time nor channel conductance was affected by PKA. The reduction in GABA receptor current by PKA was primarily due to a reduction in channel opening frequency.

LKB1, a novel serine/threonine protein kinase and potential tumour suppressor, is phosphorylated by cAMP-dependent protein kinase (PKA) and prenylated in vivo.

Peutz-Jeghers syndrome (PJS) is an autosomal dominant disease characterized by melanocytic macules, hamartomatous polyps and an increased risk for numerous cancers. The human LKB1 (hLKB1) gene encodes a serine/threonine protein kinase that is deficient in the majority of patients with PJS. The murine LKB1 (mLKB1) cDNA was isolated, sequenced and shown to produce a 2.4-kb transcript encoding a 436 amino acid protein with 90% identity with hLKB1. RNA blot and RNase-protection analysis revealed that mLKB1 mRNA is expressed in all tissues and cell lines examined. The widespread expression of LKB1 transcripts is consistent with the elevated risk of multiple cancer types in PJS patients. The predicted LKB1 protein sequence terminates with a conserved prenylation motif (Cys(433)-Lys-Gln-Gln(436)) directly downstream from a consensus cAMP-dependent protein kinase (PKA) phosphorylation site (Arg(428)-Arg-Leu-Ser(431)). The expression of enhanced green fluorescent protein (EGFP)-mLKB1 chimaeras demonstrated that LKB1 possesses a functional prenylation motif that is capable of targeting EGFP to cellular membranes. Mutation of Cys(433) to an alanine residue, but not phosphorylation by PKA, blocked membrane localization. These findings suggest that PKA does phosphorylate LKB1, although this phosphorylation does not alter the cellular localization of LKB1.

Phosphatidylinositol 3-kinase and Akt effectors mediate insulin-like growth factor-I neuroprotection in dorsal root ganglia neurons

Insulin‐like growth factor‐I (IGF‐I) protects neurons of the peripheral nervous system from apoptosis, but the underlying signaling pathways are not well understood. We studied IGF‐I mediated signaling in embryonic dorsal root ganglia (DRG) neurons. DRG neurons express IGF‐I receptors (IGF‐IR), and IGF‐I activates the phosphatidylinositol 3‐kinase (PI3K)/Akt pathway. High glucose exposure induces apoptosis, which is inhibited by IGF‐I through the PI3K/Akt pathway. IGF‐I stimulation of the PI3K/Akt pathway phosphorylates three known Akt effectors: the survival transcription factor cyclic AMP response element binding protein (CREB) and the pro‐apoptotic effector proteins glycogen synthase kinase‐3β (GSK‐3β) and forkhead (FKHR). IGF‐I regulates survival at the nuclear level through accumulation of phospho‐Akt in DRG neuronal nuclei, increased CREB‐mediated transcription, and nuclear exclusion of FKHR. High glucose increases expression of the pro‐apoptotic Bcl protein Bim (a transcriptional target of FKHR). However, IGF‐I does not regulate Bim or anti‐apoptotic Bcl‐xL protein expression levels, which suggests that IGF‐I neuroprotection is not through regulation of their expression. High glucose also induces loss of the initiator caspase‐9 and increases caspase‐3 cleavage, effects blocked by IGF‐I. These data suggest that IGF‐I prevents apoptosis in DRG neurons by regulating PI3K/Akt pathway effectors, including GSK‐3β, CREB, and FKHR, and by blocking caspase activation.

The Type II Isoform of cGMP-dependent Protein Kinase Is Dimeric and Possesses Regulatory and Catalytic Properties Distinct from the Type I Isoforms

The type I cGMP-dependent protein kinases (cGK Iα and Iβ) form homodimers (subunit Mr ∼ 76,000), presumably through conserved, amino-terminal leucine zipper motifs. Type II cGMP-dependent protein kinase (cGK II) has been reported to be monomeric (Mr ∼ 86,000), but recent cloning and sequencing of mouse brain cGK II cDNA revealed a leucine zipper motif near its amino terminus. In the present study, recombinant mouse brain cGK II was expressed, purified, and characterized. Sucrose gradient centrifugation and gel filtration chromatography were used to determine Mr values for holoenzymes of cGK Iα (168,000) and cGK II (152,500), which suggest that both are dimers. Native cGK Iα possessed significantly lower Ka values for cGMP (8-fold) and β-phenyl-1,N2-etheno-cGMP (300-fold) than did recombinant cGK II. Conversely, the Sp- and Rp-isomers of 8-(4-chloro-phenylthio)-guanosine-3′,5′-cyclic monophosphorothioate demonstrated selectivity toward cGK II in assays of kinase activation or inhibition, respectively. A peptide substrate derived from histone f2B had a 20-fold greater Vmax/Km ratio for cGK Iα than for cGK II, whereas a peptide based upon a cAMP response element binding protein phosphorylation site exhibited a greater Vmax/Km ratio for cGK II. Finally, gel filtration of extracts of mouse intestine partially resolved two cGK activities, one of which had properties similar to those demonstrated by recombinant cGK II. The combined results show that both cGK I and cGK II form homodimers but possess distinct cyclic nucleotide and substrate specificities.

A Transforming Growth Factor β-Induced Smad3/Smad4 Complex Directly Activates Protein Kinase A

ABSTRACT Transforming growth factor β (TGFβ) interacts with cell surface receptors to initiate a signaling cascade critical in regulating growth, differentiation, and development of many cell types. TGFβ signaling involves activation of Smad proteins which directly regulate target gene expression. Here we show that Smad proteins also regulate gene expression by using a previously unrecognized pathway involving direct interaction with protein kinase A (PKA). PKA has numerous effects on growth, differentiation, and apoptosis, and activation of PKA is generally initiated by increased cellular cyclic AMP (cAMP). However, we found that TGFβ activates PKA independent of increased cAMP, and our observations support the conclusion that there is formation of a complex between Smad proteins and the regulatory subunit of PKA, with release of the catalytic subunit from the PKA holoenzyme. We also found that the activation of PKA was required for TGFβ activation of CREB, induction of p21Cip1, and inhibition of cell growth. Taken together, these data indicate an important and previously unrecognized interaction between the TGFβ and PKA signaling pathways.

Phosphorylation-dependent inhibition of protein phosphatase-1 by G- substrate: A purkinje cell substrate of the cyclic GMP-dependent protein kinase

G-substrate, a specific substrate of the cGMP-dependent protein kinase, has previously been localized to the Purkinje cells of the cerebellum. We report here the isolation from mouse brain of a cDNA encoding G-substrate. This cDNA was used to localize G-substrate mRNA expression, as well as to produce recombinant protein for the characterization of G-substrate phosphatase inhibitory activity. Brain and eye were the only tissues in which a G-substrate transcript was detected. Within the brain, G-substrate transcripts were restricted almost entirely to the Purkinje cells of the cerebellum, although transcripts were also detected at low levels in the paraventricular region of the hypothalamus and the pons/medulla. Like the native protein, the recombinant protein was preferentially phosphorylated by cGMP-dependent protein kinase (K m = 0.2 μm) over cAMP-dependent protein kinase (K m = 2.0 μm). Phospho-G-substrate inhibited the catalytic subunit of native protein phosphatase-1 with an IC50 of 131 ± 27 nm. Dephospho-G-substrate was not found to be inhibitory. Both dephospho- and phospho-G-substrate were weak inhibitors of native protein phosphatase-2A1, which dephosphorylated G-substrate 20 times faster than the catalytic subunit of protein phosphatase-1. G-substrate potentiated the action of cAMP-dependent protein kinase on a cAMP-regulated luciferase reporter construct, consistent with an inhibition of cellular phosphatases in vivo. These results provide the first demonstration that G-substrate inhibits protein phosphatase-1 and suggest a novel mechanism by which cGMP-dependent protein kinase I can regulate the activity of the type 1 protein phosphatases.

Cyclic AMP- and Cyclic GMP-dependent Protein Kinases Differ in Their Regulation of Cyclic AMP Response Element-dependent Gene Transcription

The ability of cGMP-dependent protein kinases (cGKs) to activate cAMP response element (CRE)-dependent gene transcription was compared with that of cAMP-dependent protein kinases (cAKs). Although both the type Iβ cGMP-dependent protein kinase (cGKIβ) and the type II cAMP-dependent protein kinase (cAKII) phosphorylated the cytoplasmic substrate VASP (vasodilator- and A kinase-stimulatedphosphoprotein) to a similar extent, cyclic nucleotide regulation of CRE-dependent transcription was at least 10-fold higher in cAKII-transfected cells than in cGKIβ-transfected cells. Overexpression of each kinase in mammalian cells resulted in a cytoplasmic localization of the unactivated enzyme. As reported previously, the catalytic (C) subunit of cAKII translocated to the nucleus following activation by 8-bromo-cyclic AMP. However, cGKIβ did not translocate to the nucleus upon activation by 8-bromo-cyclic GMP. Replacement of an autophosphorylated serine (Ser79) of cGKIβ with an aspartic acid resulted in a mutant kinase with constitutive kinase activity in vitroand in vivo. The cGKIβS79D mutant localized to the cytoplasm and was only a weak activator of CRE-dependent gene transcription. However, an amino-terminal deletion mutant of cGKIβ was found in the nucleus as well as the cytoplasm and was a strong activator of CRE-dependent gene transcription. These data suggest that the inability of cGKs to translocate to the nucleus is responsible for the differential ability of cAKs and cGKs to activate CRE-dependent gene transcription and that nuclear redistribution of cGKs is not required for NO/cGMP regulation of gene transcription.

Characterization of PKIγ, a Novel Isoform of the Protein Kinase Inhibitor of cAMP-dependent Protein Kinase

Attempts to understand the physiological roles of the protein kinase inhibitor (PKI) proteins have been hampered by a lack of knowledge concerning the molecular heterogeneity of the PKI family. The PKIγ cDNA sequence determined here predicted an open reading frame of 75 amino acids, showing 35% identity to PKIα and 30% identity to PKIβ1. Residues important for the high affinity of PKIα and PKIβ1 as well as nuclear export of the catalytic (C) subunit of cAMP-dependent protein kinase were found to be conserved in PKIγ. Northern blot analysis showed that a 1.3-kilobase PKIγ message is widely expressed, with highest levels in heart, skeletal muscle, and testis. RNase protection analysis revealed that in most tissues examined PKIγ is expressed at levels equal to or higher than the other known PKI isoforms and that in several mouse-derived cell lines, PKIγ is the predominant PKI message. Partial purification of PKI activities from mouse heart by DEAE ion exchange chromatography resolved two major inhibitory peaks, and isoform-specific polyclonal antibodies raised against recombinant PKIα and PKIγ identified these inhibitory activities to be PKIα and PKIγ. A comparison of inhibitory potencies of PKIα and PKIγ expressed inEscherichia coli revealed that PKIγ was a potent competitive inhibitor of Cα phosphotransferase activity in vitro (K i = 0.44 nm) but is 6-fold less potent than PKIα (K i = 0.073 nm). Like PKIα, PKIγ was capable of blocking the nuclear accumulation of Flag-tagged C subunit in transiently transfected mammalian cells. Finally, the murine PKIγ gene was found to overlap the murine adenosine deaminase gene on mouse chromosome 2. These results demonstrate that PKIγ is a novel, functional PKI isoform that accounts for the previously observed discrepancy between PKI activity and PKI mRNA levels in several mammalian tissues.

Negative regulation of Yap during neuronal differentiation

Regulated proliferation and cell cycle exit are essential aspects of neurogenesis. The Yap transcriptional coactivator controls proliferation in a variety of tissues during development, and this activity is negatively regulated by kinases in the Hippo signaling pathway. We find that Yap is expressed in mitotic mouse retinal progenitors and it is downregulated during neuronal differentiation. Forced expression of Yap prolongs proliferation in the postnatal mouse retina, whereas inhibition of Yap by RNA interference (RNAi) decreases proliferation and increases differentiation. We show Yap is subject to post-translational inhibition in the retina, and also downregulated at the level of mRNA expression. Using a cell culture model, we find that expression of the proneural basic helix-loop-helix (bHLH) transcription factors Neurog2 or Ascl1 downregulates Yap mRNA levels, and simultaneously inhibits Yap protein via activation of the Lats1 and/or Lats2 kinases. Conversely, overexpression of Yap prevents proneural bHLH proteins from initiating cell cycle exit. We propose that mutual inhibition between proneural bHLH proteins and Yap is an important regulator of proliferation and cell cycle exit during mammalian neurogenesis.

The cyclic AMP‐dependent protein kinase catalytic subunit selectively enhances calcium currents in rat nodose neurones.

1. The whole‐cell variation of the patch clamp technique was used to study the effect of the purified catalytic subunit of the cyclic AMP‐dependent protein kinase (A kinase catalytic subunit: AK‐C) on the calcium current components of acutely dissociated rat nodose ganglion neurones. 2. The transient low‐threshold calcium current component (T) was stable during whole‐cell recording. In contrast, currents containing the transient high‐threshold (N) and slowly inactivating high‐threshold (L) current components declined steadily after stabilization of the currents during the first 5‐7 min of recording. When AK‐C was included in the recording pipette at physiological concentrations (50 micrograms/ml, approximately 1 microM), currents containing the N‐ and L‐components increased in magnitude beginning 7‐9 min after patch rupture, but there was no effect on the isolated T‐current. The current‐voltage relation of the T‐current component was similar to controls, but the current‐voltage relation for the N‐ and L‐current components was shifted slightly to more depolarized clamp potentials (Vc), approximately 10 mV. 3. The effect of AK‐C on currents containing the N‐ and L‐currents was concentration dependent. There was no effect of 0.1 microgram/ml AK‐C, the lowest concentration tested. Currents evoked from holding potentials (Vh) = ‐80 mV increased 5‐10% during a 20 min recording in the presence of 1 microgram/ml AK‐C and 30‐35% in the presence of 50 micrograms/ml AK‐C. In contrast, currents evoked from Vh = ‐40 mV increased 5‐10% in the presence of either 1 or 50 micrograms/ml AK‐C. The increase in current magnitude was associated with an increased rate of current inactivation and was evident particularly in currents evoked from Vh = ‐80 mV. 4. These effects were blocked by prior incubation of AK‐C (1 microgram/ml) with a specific peptide inhibitor (protein kinase inhibitor peptide, PKIP; 0.2 mg/ml). 5. We evoked calcium currents using very long (1 s) voltage commands and modelled the traces using a multiexponential function in order to determine the effects of AK‐C on the N‐ and L‐current components. The (curve‐fitted) N‐ and L‐current components each declined approximately 50% during a 20 min recording in control neurones.(ABSTRACT TRUNCATED AT 250 WORDS)