Howard Jaffe

Inhibition of T-type voltage-gated calcium channels by a new scorpion toxin.

The biophysical properties of T-type voltage-gated calcium channels are well suited to pacemaking and to supporting calcium flux near the resting membrane potential in both excitable and non-excitable cells. We have identified a new scorpion toxin (kurtoxin) that binds to the α1G T-type calcium channel with high affinity and inhibits the channel by modifying voltage-dependent gating. This toxin distinguishes between α1G T-type calcium channels and other types of voltage-gated calcium channels, including α 1A, α1B, α1C and α1E. Like the other α-scorpion toxins to which it is related, kurtoxin also interacts with voltage-gated sodium channels and slows their inactivation. Kurtoxin will facilitate characterization of the subunit composition of T-type calcium channels and help determine their involvement in electrical and biochemical signaling.

Role of a pineal cAMP-operated arylalkylamine N-acetyltransferase/14-3-3-binding switch in melatonin synthesis.

The daily rhythm in melatonin levels is controlled by cAMP through actions on the penultimate enzyme in melatonin synthesis, arylalkylamine N-acetyltransferase (AANAT; serotonin N-acetyltransferase, EC 2.3.1.87). Results presented here describe a regulatory/binding sequence in AANAT that encodes a cAMP-operated binding switch through which cAMP-regulated protein kinase-catalyzed phosphorylation [RRHTLPAN → RRHpTLPAN] promotes formation of a complex with 14-3-3 proteins. Formation of this AANAT/14-3-3 complex enhances melatonin production by shielding AANAT from dephosphorylation and/or proteolysis and by decreasing the Km for 5-hydroxytryptamine (serotonin). Similar switches could play a role in cAMP signal transduction in other biological systems.

Cyclin‐dependent kinase 5 prevents neuronal apoptosis by negative regulation of c‐Jun N‐terminal kinase 3

Cyclin‐dependent kinase 5 (cdk5) is a serine/threonine kinase activated by associating with its neuron‐specific activators p35 and p39. Analysis of cdk5−/− and p35−/− mice has demonstrated that both cdk5 and p35 are essential for neuronal migration, axon pathfinding and the laminar configuration of the cerebral cortex, suggesting that the cdk5–p35 complex may play a role in neuron survival. However, the targets of cdk5 that regulate neuron survival are unknown. Here, we show that cdk5 directly phosphorylates c‐Jun N‐terminal kinase 3 (JNK3) on Thr131 and inhibits its kinase activity, leading to reduced c‐Jun phosphorylation. Expression of cdk5 and p35 in HEK293T cells inhibits c‐Jun phosphorylation induced by UV irradiation. These effects can be restored by expression of a catalytically inactive mutant form of cdk5. Moreover, cdk5‐deficient cultured cortical neurons exhibit increased sensitivity to apoptotic stimuli, as well as elevated JNK3 activity and c‐Jun phosphorylation. Taken together, these findings show that cdk5 may exert its role as a key element by negatively regulating the c‐Jun N‐terminal kinase/stress‐activated protein kinase signaling pathway during neuronal apoptosis.

The Parkinson’s disease kinase LRRK2 autophosphorylates its GTPase domain at multiple sites

Mutations in leucine-rich repeat kinase 2 (LRRK2) are a common cause of inherited Parkinsons disease (PD). The protein is large and complex, but pathogenic mutations cluster in a region containing GTPase and kinase domains. LRRK2 can autophosphorylate in vitro within a dimer pair, although the significance of this reaction is unclear. Here, we mapped the sites of autophosphorylation within LRRK2 and found several potential phosphorylation sites within the GTPase domain. Using mass spectrometry, we found that Thr1343 is phosphorylated and, using kinase dead versions of LRRK2, show that this is an autophosphorylation site. However, we also find evidence for additional sites in the GTPase domain and in other regions of the protein suggesting that there may be multiple autophosphorylation sites within LRRK2. These data suggest that the kinase and GTPase activities of LRRK2 may exhibit complex autoregulatory interdependence.

The familial Mediterranean fever protein, pyrin, is cleaved by caspase-1 and activates NF-κB through its N-terminal fragment

Familial Mediterranean fever (FMF) is an autoinflammatory disease caused by mutations in MEFV, which encodes a 781-amino acid protein denoted pyrin. We have previously shown that pyrin regulates caspase-1 activation and IL-1beta production through interaction of its N-terminal PYD motif with the ASC adapter protein, and also modulates IL-1beta production by interaction of its C-terminal B30.2 domain with the catalytic domains of caspase-1. We now asked whether pyrin might itself be a caspase-1 substrate, and found that pyrin is cleaved by caspase-1 at Asp330, a site remote from the B30.2 domain. Pyrin variants harboring FMF-associated B30.2 mutations were cleaved more efficiently than wild-type pyrin. The N-terminal cleaved fragment interacted with the p65 subunit of NF-kappaB and with IkappaB-alpha through its 15-aa bZIP basic domain and adjacent sequences, respectively, and translocated to the nucleus. The interaction of the N-terminal fragment with p65 enhanced entrance of p65 into the nucleus. The interaction of N-terminal pyrin with IkappaB-alpha induced calpain-mediated degradation of IkappaB-alpha, thus potentiating NF-kappaB activation. Absolute and relative quantities of cleaved pyrin and IkappaB-alpha degradation products were substantially increased in leukocytes from FMF patients compared with healthy controls. Our data support a new pyrin/caspase-1 pathway for NF-kappaB activation.

Purification and Cloning of the GTP Cyclohydrolase I Feedback Regulatory Protein, GFRP

The activity of GTP cyclohydrolase I, the initial enzyme of the de novo pathway for biosynthesis of tetrahydrobiopterin, the cofactor required for aromatic amino acid hydroxylations and nitric oxide synthesis, is sensitive to end-product feedback inhibition by tetrahydrobiopterin. This inhibition by tetrahydrobiopterin is mediated by the GTP cyclohydrolase I feedback regulatory protein GFRP, previously named p35 (Harada, T., Kagamiyama, H., and Hatakeyama, K. (1993) Science 260, 1507-1510), and L-phenylalanine specifically reverses the tetrahydrobiopterin-dependent inhibition. As a first step in the investigation of the physiological role of this unique mechanism of regulation, a convenient procedure has been developed to co-purify to homogeneity both GTP cyclohydrolase I and GFRP from rat liver. GTP cyclohydrolase I and GFRP exist in a complex which can be bound to a GTP-affinity column from which GTP cyclohydrolase I and GFRP are separately and selectively eluted. GFRP is dissociated from the GTP agarose-bound complex with 0.2 M NaCl, a concentration of salt which also effectively blocks the tetrahydrobiopterin-dependent inhibitory activity of GFRP. GTP cyclohydrolase I is then eluted from the GTP-agarose column with GTP. Both GFRP and GTP cyclohydrolase I were then purified separately to near homogeneity by sequential high performance anion exchange and gel filtration chromatography. GFRP was found to have a native molecular mass of 20 kDa and consist of a homodimer of 9.5-kDa subunits. Based on peptide sequences obtained from purified GFRP, oligonucleotides were synthesized and used to clone a cDNA from a rat liver cDNA library by polymerase chain reaction-based methods. The cDNA contained an open reading frame that encoded a novel protein of 84 amino acids (calculated molecular mass 9665 daltons). This protein when expressed in Escherichia coli as a thioredoxin fusion protein had tetrahydrobiopterin-dependent GTP cyclohydrolase I inhibitory activity. Northern blot analysis indicated the presence of an 0.8-kilobase GFRP mRNA in most rat tissues, the amounts generally correlating with levels of GTP cyclohydrolase I and tetrahydrobiopterin. Thus, mRNA levels were relatively high in liver and kidney and somewhat lower in testis, heart, brain, and lung. These results suggest that GFRP is widely expressed and may play a role in regulating not only phenylalanine metabolism in the liver, but also the production of biogenic amine neurotransmitters as well as nitric oxide synthesis.

Neuronal Cyclin‐Dependent Kinase‐5 Phosphorylation Sites in Neurofilament Protein (NF‐H) Are Dephosphorylated by Protein Phosphatase 2A

Abstract: Neurofilament (NF) protein [high molecular mass (NF‐H)] is extensively phosphorylated in vivo. The phosphorylation occurs mainly in its characteristic KSP (Lys‐Ser‐Pro) repeat motifs. There are two major types of KSP motifs in the NF‐H tail domain: KSPXKX and KSPXXX. Recent studies by two different laboratories have demonstrated the presence of a cdc2‐like kinase [cyclin‐dependent kinase‐5 (cdk5)] in nervous tissue that selectively phosphorylates KSPXKX and XS/TXK motifs in NF‐H and lysine‐rich histone (H1). This article describes the identification of phosphatases dephosphorylating three different substrates: histone (H1), NF‐H in a NF preparation, and a bacterially expressed C‐terminal tail domain of NF‐H, each containing KSPXKX repeats phosphorylated in vitro by cdk5. Among various phosphatases identified, protein phosphatase (PP) 2A from rabbit skeletal muscle appeared to be the most effective phosphatase in in vitro assays. Three phosphatase activity peaks—P1, P2, and P3—were partially purified from frozen rat spinal cord by ion exchange and size exclusion column chromatography and then characterized on the basis of biochemical, pharmacological, and immunochemical studies. One of the three peaks was identified as PP2A, whereas the others were mixtures of both PP2A and PP1. These three peaks could dephosphorylate cdk5‐phosphorylated 32P‐histone (H1), 32P‐NF‐H in the NF preparation, and 32P‐NF‐H tail fusion protein. These studies suggest the involvement of PP2A or a PP2A‐like activity in the regulation of the phosphorylation state of KSPXKX motifs in NF‐H.

Molecular characterization of a neuronal-specific protein that stimulates the activity of Cdk5

Abstract: Cyclin‐dependent kinase, Cdk5, has been identified in neural tissue in connection with neurofilament and τ protein phosphorylation. This report describes the characterization of a 62‐kDa protein that copurifies with Cdk5 from rat spinal cord homogenates. Dissociation of the protein from neural Cdk5 is concomitant with a reversible loss in kinase activity. Amino acid sequence information from tryptic peptide fragments was used to clone the complementary DNA from rat brain. A single full‐length cDNA was characterized coding for a 67.5‐kDa protein (p67). Exogenously expressed p67 stimulated Cdk5 kinase activity in vitro in a dose‐dependent manner and when presented as an affinity matrix, selectively adsorbed Cdk5 from a cleared rat brain homogenate. In situ hybridization analysis of E18 rat embryos and adult rat brain demonstrated that p67 transcript expression is restricted to neural tissue. Immunohistochemical staining with an amino‐terminal peptide‐specific antibody further indicated that p67 is exclusively expressed in neurons. Localization in vivo and in cultured rat hippocampal neurons showed that p67 is highly enriched in axons. We propose that p67, by virtue of its regulation of Cdk5, participates in the dynamics of axonal architecture through the modulation of phosphorylation of cytoskeletal components.

Identification of Endogenously Phosphorylated KSP Sites in the High-Molecular-Weight Rat Neurofilament Protein

Abstract: The high‐molecular‐weight neurofilament protein (NF‐H) is highly phosphorylated in vivo, with estimates as high as 16–51 mol of Pi/mol of protein. Most of the phosphorylation sites are thought to be located on Ser residues in multiple KSP repeats, in the carboxy‐terminal tail region of the molecule. Because the extent and site‐specific patterns of tail domain phosphorylation are believed to modulate neurofilament structure and function, it becomes essential to identify the endogenous sites of phosphorylation. In this study, we have used selective proteolytic cleavage procedures, Pi determinations, microsequencing, and mass‐spectral analysis to determine the endogenously phosphorylated sites in the NF‐H tail isolated from rat spinal cord. Twenty Ser residues in NF‐H carboxy‐terminal tail were analyzed; nine of these, all located in KSP repeats, were phosphorylated. No detectable phosphorylation could be identified in any of the 11 “non‐KSP” Ser residues that were examined. KSPXKX, KSPXXX, and KSPXXK motifs were found to be phosphorylated. In addition, a 27‐kDa KSP‐rich domain, containing 43 virtually uninterrupted KSPXXX repeats, was isolated from the tail domain and found to contain between 30 and 35 mol of Pi/mol of protein. This domain appeared to be highly resistant to endoproteinase Glu‐C digestion, although it contains a large number of glutamate residues. It could be proteolyzed, however, after dephosphorylation. This suggests that phosphorylation of the tail domain may contribute to neurofilament stability in vivo. A neuronal‐derived protein kinase that specifically phosphorylates only KSPXKX motifs in neurofilaments has been reported. The presence of extensively phosphorylated KSPXXX repeats in NF‐H in vivo suggests the existence of yet another, unidentified kinase(s) with specificity for KSPXXX motifs.

Phosphorylation state of the native high-molecular-weight neurofilament subunit protein from cervical spinal cord in sporadic amyotrophic lateral sclerosis

The intraneuronal aggregation of phosphorylated high‐molecular‐weight neurofilament protein (NFH) in spinal cord motor neurons is considered to be a key pathological marker of amyotrophic lateral sclerosis (ALS). In order to determine whether this observation is due to the aberrant or hyper‐phosphorylation of NFH, we have purified and characterized NFH from the cervical spinal cords of ALS patients and controls. We observed no differences between ALS and normal controls in the physicochemical properties of NFH in Triton X‐100 insoluble protein fractions, with respect to migration patterns on 2D‐iso electrofocusing (IEF) gels, the rate of Escherichia coli alkaline phosphatase mediated dephosphorylation, or the rate of calpain‐mediated proteolysis. The rate of calpain‐mediated proteolysis was unaffected by either exhaustive NFH dephosphorylation or by the addition of calmodulin to the reaction. Phosphopeptides and the phosphorylated motifs characterized by liquid chromatography tandem mass spectroscopy (LC/MS/MS) analysis demonstrated that all the phosphorylated residues found in ALS NFH were also found to be phosphorylated in normal human NFH samples. Hence, we have observed no difference in the physicochemical properties of normal and ALS NFH extracted from cervical spinal cords, suggesting that the perikaryal aggregation of highly phosphorylated NF in ALS neurons reflects the aberrant somatotopic localization of normally phosphorylated NFH.