scholarly journals Differential inhibition of nerve growth factor responses by purine analogues: correlation with inhibition of a nerve growth factor-activated protein kinase.

1989 ◽  
Vol 109 (5) ◽  
pp. 2395-2403 ◽  
Author(s):  
C Volonté ◽  
A Rukenstein ◽  
D M Loeb ◽  
L A Greene
Keyword(s):  
Nerve Growth Factor ◽  
Growth Factor ◽  
Protein Kinase ◽  
Protein Kinases ◽  
Pc12 Cells ◽  
Ornithine Decarboxylase ◽  
Decarboxylase Activity ◽  
Purine Analogues ◽  
Nerve Growth ◽  
Protein Kinase N

Purine analogues were used in this study to dissect specific steps in the mechanism of action of nerve growth factor (NGF). Protein kinase N (PKN) is an NGF-activated serine protein kinase that is active in the presence of Mn++. The activity of PKN was inhibited in vitro by purine analogues, the most effective of which was 6-thioguanine (apparent Ki = 6 microM). Several different criteria indicated that 6-thioguanine is not a general inhibitor of protein kinases and that it is relatively specific for PKN. For instance, it did not affect protein kinases A or C and was without effect on the overall level and pattern of protein phosphorylation by either intact or broken PC12 cells. Since purine analogues rapidly and effectively enter cells, they were also assessed for their actions on both transcription-dependent and -independent responses of PC12 cells to NGF. NGF-promoted neurite regeneration was reversibly suppressed by the analogues and at concentrations very similar to those that inhibit PKN. Comparable concentrations of the analogues also blocked NGF-stimulated induction of ornithine decarboxylase activity. In contrast to its inhibition of neurite regeneration and ornithine decarboxylase induction, 6-thioguanine did not suppress NGF-dependent induction of c-fos mRNA expression. Thus, purine analogues such as 6-thioguanine appear capable of differentially suppressing some, but not other actions of NGF. These findings suggest the presence of multiple pathways in the NGF mechanism and that these can be dissected with purine analogues. Moreover, these data are compatible with a role for protein kinase N in certain of these pathways.

scholarly journals Association of a purine-analogue-sensitive protein kinase activity with p75 nerve growth factor receptors.

1993 ◽  
Vol 4 (1) ◽  
pp. 71-78 ◽  
Author(s):  
C Volonté ◽  
A H Ross ◽  
L A Greene
Keyword(s):  
Nerve Growth Factor ◽  
Growth Factor ◽  
Protein Kinase ◽  
Pc12 Cells ◽  
Kinase Activity ◽  
Cellular Systems ◽  
Protein Kinase Activity ◽  
Purine Analogues ◽  
Nerve Growth ◽  
Purine Analogue

Purine analogues are protein kinase inhibitors, and they block with varying potency and specificity certain of the biological actions of nerve growth factor (NGF). The analogue 6-thioguanine (6-TG) has been shown to inhibit with high specificity protein kinase N (PKN), a serine/threonine protein kinase activated by NGF in several cellular systems. In the present work, immunoprecipitates of p75 NGF receptors from PC12 cells (+/-NGF treatment) were assayed for protein kinase activity using the substrate myelin basic protein under phosphorylating conditions optimal for PKN and in the presence or absence of purine analogues. An NGF-inducible activity was detected, and approximately 80% was inhibited by purine analogues. This activity was maximally stimulated by NGF within 5-10 min, partially decreased by 60 min, and returned to basal levels after 15 h of NGF treatment. The analogue 6-TG inhibited the NGF-inducible p75-associated kinase activity with an IC50 in the range of 15-35 microM. In mutant PC12 nnr-5 cells that lack the Trk NGF receptor, the purine-analogue-sensitive p75-associated kinase activity was not inducible by NFG. In normal PC12 cells, cyclic AMP analogues and epidermal growth factor failed to induce the same activity. Application of either 2-aminopurine or 6-TG to intact cells only slightly inhibit the NGF-dependent induction of the purine-analogue-inhibited p75-associated kinase activity. This activity shares many similarities but also displays some significant differences with cytosolic PKN. Our findings therefore indicate the association of a purine-analogue-sensitive protein kinase with p75 NGF receptors.

scholarly journals Nerve Growth Factor Stimulates Multisite Tyrosine Phosphorylation and Activation of the Atypical Protein Kinase C's via a src Kinase Pathway

2001 ◽  
Vol 21 (24) ◽  
pp. 8414-8427 ◽  
Author(s):  
Marie W. Wooten ◽  
Michel L. Vandenplas ◽  
M. Lamar Seibenhener ◽  
Thangiah Geetha ◽  
Maria T. Diaz-Meco
Keyword(s):  
Nerve Growth Factor ◽  
Growth Factor ◽  
Protein Kinase ◽  
Tyrosine Phosphorylation ◽  
Pc12 Cells ◽  
Catalytic Domain ◽  
Src Kinase ◽  
Dependent Manner ◽  
Nerve Growth ◽  
Atypical Protein Kinase

ABSTRACT Atypical protein kinase C (PKC) isoforms are required for nerve growth factor (NGF)-initiated differentiation of PC12 cells. In the present study, we report that PKC-ι becomes tyrosine phosphorylated in the membrane coincident with activation posttreatment with nerve growth factor. Tyrosine phosphorylation and activation of PKC-ι were inhibited in a dose-dependent manner by both PP2 and K252a, src and TrkA kinase inhibitors. Purified src was observed to phosphorylate and activate PKC-ι in vitro. In PC12 cells deficient in src kinase activity, both NGF-induced tyrosine phosphorylation and activation of PKC-ι were also diminished. Furthermore, we demonstrate activation of src by NGF along with formation of a signal complex including the TrkA receptor, src, and PKC-ι. Recruitment of PKC-ι into the complex was dependent on the tyrosine phosphorylation state of PKC-ι. The association of src and PKC-ι was constitutive but was enhanced by NGF treatment, with the src homology 3 domain interacting with a PXXP sequence within the regulatory domain of PKC-ι (amino acids 98 to 114). Altogether, these findings support a role for src in regulation of PKC-ι. Tyrosine 256, 271, and 325 were identified as major sites phosphorylated by src in the catalytic domain. Y256F and Y271F mutations did not alter src-induced activation of PKC-ι, whereas the Y325F mutation significantly reduced src-induced activation of PKC-ι. The functional relevance of these mutations was tested by determining the ability of each mutant to support TRAF6 activation of NF-κB, with significant impairment by the Y325F PKC-ι mutant. Moreover, when the Y352F mutant was expressed in PC12 cells, NGF's ability to promote survival in serum-free media was reduced. In summary, we have identified a novel mechanism for NGF-induced activation of atypical PKC involving tyrosine phosphorylation by c-Src.

scholarly journals Nerve growth factor employs multiple pathways to induce primary response genes in PC12 cells.

1992 ◽  
Vol 3 (3) ◽  
pp. 363-371 ◽  
Author(s):  
A Batistatou ◽  
C Volonté ◽  
L A Greene
Keyword(s):  
Nerve Growth Factor ◽  
Growth Factor ◽  
Pc12 Cells ◽  
Primary Response ◽  
Purine Analogues ◽  
Signaling Mechanism ◽  
Nerve Growth ◽  
Primary Response Genes ◽  
Run Off ◽  
Ngf Signaling

Nerve growth factor (NGF) leads to neuronal differentiation of PC12 cells and promotes their survival in serum-free medium. Past studies have shown that purine analogues block some of the effects of NGF but not others and thus that they can be used to dissect the mechanistic pathways of its action. In the present work we used 2-aminopurine (2-AP) and 6-thioguanine (6-TG) to examine whether NGF causes activation of primary response genes through a single signaling pathway or via multiple pathways. Northern blot analysis and nuclear run-off transcription assays were used to assess the activation of c-fos, c-jun, TIS1, TIS8, and TIS11 after exposure of PC12 cells to NGF in the presence or absence of 2-AP and 6-TG. Our findings indicate that NGF appears to employ at least three distinct pathways to induce early genes in PC12 cells. This suggests that the NGF signaling mechanism diverges at an early point after interaction of NGF with its receptor.

Nerve growth factor enhances the level of the protein kinase C substrate B-50 in pheochromocytoma PC12 cells

1986 ◽  
Vol 139 (2) ◽  
pp. 644-651 ◽  
Author(s):  
C.O.M. Van Hooff ◽  
P.N.E. De Graan ◽  
J. Boonstra ◽  
A.B. Oestreicher ◽  
M.H. Schmidt-Michels ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Nerve Growth Factor ◽  
Growth Factor ◽  
Protein Kinase ◽  
Pc12 Cells ◽  
Nerve Growth ◽  
Kinase C ◽  
Pheochromocytoma Pc12 ◽  
Pheochromocytoma Pc12 Cells
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