Purine analogs inhibit nerve growth factor-promoted neurite outgrowth by sympathetic and sensory neurons

Greene, Lloyd A.; Volonté, Cinzia; Chalazonitis, Alcmène

doi:10.1523/jneurosci.10-05-01479.1990

Cited by 61 publications

(55 citation statements)

References 25 publications

(23 reference statements)

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“…1A), two cycles of reversed-phase HPLC, and SDS/PAGE. Throughout, a protein band of Ϸ50 kDa exhibited the properties previously described for the kinase of interest, i.e., a capacity to phosphorylate histone protein HF1 in a 6-TG-sensitive, Ca 2ϩ -, Mg 2ϩ -, and Mn 2ϩ -insensitive fashion (11)(12)(13)(14). Proteolytic fragments of the purified kinase, when sequenced by LC-MS, exactly matched portions of human Mst3 (15).…”

Section: Resultsmentioning

confidence: 74%

“…The observation that 6-TG blocks axon outgrowth in primary neurons (9,12) suggested to us that the kinase it inhibits might be widely expressed in the brain. We prepared a high-speed supernatant fraction from neonatal bovine cortex and applied this material to a sulfopropyl-Sepharose column, which was previously shown to yield a partial purification of a 6-TG-sensitive kinase from PC12 pheochromocytoma cells (13).…”

Section: Resultsmentioning

confidence: 98%

“…The purine analog 6-thioguanine (6-TG), on the other hand, blocks outgrowth induced by neurotrophic factors (9,11,12), and this effect is paralleled by the inhibition of a previously unidentified 45-50 kDa serine-threonine protein kinase (13). Inosine competitively reverses the inhibitory effects of 6-TG on outgrowth (7,9), suggesting that it may act as an agonist of the 6-TG-sensitive kinase.…”

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confidence: 99%

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Mst3b, a purine-sensitive Ste20-like protein kinase, regulates axon outgrowth

Irwin

Li²,

O'Toole³

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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The growth of axons is fundamental to the development and repair of brain circuitry. We show here that Mst3b, a neuron-specific homolog of the yeast kinase Ste20, is critical for axon outgrowth. Mst3b is activated in response to trophic factors, and suppressing its expression (via siRNAs) or its function (by a dominant-negative mutant) blocks axon outgrowth. Inosine, a purine nucleoside that stimulates axon outgrowth, activates Mst3b kinase activity, whereas 6-thioguanine, a purine analog that blocks outgrowth, inhibits the activity of this kinase. These findings place Mst3b as a key regulator of axon outgrowth and help explain the purine sensitivity of this process.A xon outgrowth begins shortly after neurons undergo final cell division, and it ends when axon terminals reach their appropriate targets. Although this process is not normally reactivated in the mature CNS after injury, some degree of CNS regeneration has been achieved experimentally by altering neurons' intrinsic growth state and by counteracting inhibitory signals associated with myelin, the perineuronal net, or a glial scar (1-5). Thus, understanding the mechanisms that underlie axon growth not only affords insight into how the brain's circuitry develops but may also enable us to improve functional outcome after stroke, trauma, or neurodegenerative disorders.The purine nucleoside inosine stimulates axon outgrowth in certain types of neurons in culture and in vivo after CNS injury (6-10). The purine analog 6-thioguanine (6-TG), on the other hand, blocks outgrowth induced by neurotrophic factors (9,11,12), and this effect is paralleled by the inhibition of a previously unidentified 45-50 kDa serine-threonine protein kinase (13). Inosine competitively reverses the inhibitory effects of 6-TG on outgrowth (7, 9), suggesting that it may act as an agonist of the 6-TG-sensitive kinase. We now identify the purine-sensitive kinase as mammalian Ste20-like protein kinase-3b (Mst3b) and show that it is a key regulator of axon outgrowth.

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Section: Resultsmentioning

confidence: 74%

Section: Resultsmentioning

confidence: 98%

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confidence: 99%

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Mst3b, a purine-sensitive Ste20-like protein kinase, regulates axon outgrowth

Irwin

Li²,

O'Toole³

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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“…Because the PC12 cell line represents an excellent model system for investigating the mode of action of NGF in a ho- mogeneous cell population (Greene andTischler, 1976, 1982;Greene, 1978;Greene et al, 1990), the data presented here provide a basis for studying the proposed role of extracellular protein phosphorylation systems in molecular mechanisms underlying NGF-induced neuronal differentiation in general and neuritogenesis in particular (Ehrlich et a]., 1986(Ehrlich et a]., , 1990Tsuji et al, 1988;Nagashima et al, 1991). The criteria outlined for the identification of an ecto-enzyme (Karnovsky, 1986) and the special requirements for identifying ecto-protein kinase activity (Ehrlich et al, 1990) have been fulfilled in the present studies with PC 12 cells.…”

Section: Discussionmentioning

confidence: 99%

Ecto‐Protein Kinase and Surface Protein Phosphorylation in PC12 Cells: Interactions with Nerve Growth Factor

Pawłowska

Hogan

Kornecki

et al. 1993

Journal of Neurochemistry

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Abstract:The phosphorylation of surface proteins by ectoprotein kinase has been proposed to play a role in mechanisms underlying neuronal differentiation and their responsiveness to nerve growth factor (NGF). PC 12 clones represent an optimal model for investigating the mode of action of NGF in a homogeneous cell population. In the present study we obtained evidence that PC12 cells possess ectoprotein kinase and characterized the endogenous phosphorylation of its surface protein substrates. PC12 cells maintained in a chemically defined medium exhibited phosphorylation of proteins by [y-32P]ATP added to the medium at time points preceding the intracellular phosphorylation of proteins in cells labeled with 32P,. This activity WBS abolished by adding apyrase or trypsin to the medium but was not sensitive to addition of an excess of unlabeled P,. As also expected from ecto-protein kinase activity, PC 12 cells catalyzed the phosphorylation of an exogenous protein substrate added to the medium, dephospho-a-casein, and this activity competed with the endogenous phosphorylation for extracellular ATP. Based on these criteria, three protein components migrating in sodium dodecyl sulfate gels with apparent molecular weights of 105K, 39K, and 20K were identified as e.wIusive substrates of ecto-protein kinase in PC12 cells. Of the phosphate incorporated into these proteins from extracellular ATP, 75-87% was found in phosphothreonine. The phosphorylation of the 39K protein by ecto-protein kinase did not require Mg2+, implicating this activity in the previously demonstrated regulation of Ca2+-dependent, high-affinity norepinephrine uptake in PC 12 cells by extracellular ATP. The protein kinase inhibitor K-252a inhibited both intra-and extracellular protein phosphorylation in intact PC 12 cells. Its hydrophilic analogue K-252b, had only minimal effects on intracellular protein phosphorylation but readily inhibited the phosphorylation of specific substrates of ecto-protein kinase in PC12 cells incubated with extracellular ATP, suggesting the involvement of ecto-protein kinase in the reported inhibition of NGF-induced neurite extension by K-252b. Preincubation of PC12 cells with 50 ng/ml of NGF for 5 min stimulated the activity of ecto-protein kinase toward all its endogenous substrates. Exposure of PC12 cells to the same NGF concentration for 3 days revealed another substrate of ecto-protein kinase, a 53K protein, whose surface phosphorylation is expressed only after NGF-induced neuronal differentiation. In the concentration range (10-100 p h f ) at which 6-thioguanine blocked NGF-promoted neurite outgrowth in PC12 cells, 6-thioguanine effectively inhibited the phosphorylation of specific proteins by ecto-protein kinase. This study provides the basis for continued investigation of the involvement of ecto-protein kinase and its surface protein substrates in neuronal differentiation, neuritogenesis, and synaptogenesis. Key Words: Protein phosphorylationEcto-protein kinase-Surface phosphoproteins-ExtracelMar ATP-Nerve growth factor...

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“…First, it is known that different molecular cascades are involved in NGF-dependent neurite outgrowth and NGFdependent neuronal survival of SC and sensory neurons (Greene et al, 1990). It is conceivable that similar alternative pathways are differentially activated by the chimeric molecules that display BDNF-dependent neurite outgrowth but no BDNF-dependent neuronal survival.…”

Section: Mutagenesismentioning

confidence: 99%

Homolog‐Scanning Mutagenesis

2004

Encyclopedic Dictionary of Genetics, Genomics and Proteomics

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Purine analogs inhibit nerve growth factor-promoted neurite outgrowth by sympathetic and sensory neurons

Cited by 61 publications

References 25 publications

Mst3b, a purine-sensitive Ste20-like protein kinase, regulates axon outgrowth

Mst3b, a purine-sensitive Ste20-like protein kinase, regulates axon outgrowth

Ecto‐Protein Kinase and Surface Protein Phosphorylation in PC12 Cells: Interactions with Nerve Growth Factor

Homolog‐Scanning Mutagenesis

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