Pub Date : 2014-03-01DOI: 10.4236/cellbio.2014.31001
Alana Doonachar, Alan R Schoenfeld
Atypical PKC (aPKC) plays a role in establishing cell polarity and has been indicated in neuronal differentiation and polarization, including neurite formation in rat pheochromocytoma PC12 cells, albeit by unclear mechanisms. Here, the role of the aPKC isoform, PKC iota (PKCι), in the early neuronal differentiation of PC12 cells was investigated. NGF-treated PC12 cells with stably expressed exogenous wild-type PKCι showed decreased expression of a neuroendocrine marker, increased expression of a neuronal marker, and increased neurite formation. Stable expression of a kinase- inactive PKCι, but not constitutively active PKCι lacking a regulatory domain, had similar although less potent effects. Pharmacological inhibition of endogenous aPKC kinase activity in parental PC12 cells did not inhibit neurite formation, suggesting that some of the observed effects of PKCι expression on neuronal differentiation are kinase- independent. Interestingly, exogenous expression of wild-type and kinase-inactive PKCι had little effect on overall PKCι activity, but caused a decrease in PKC zeta (PKCζ) kinase activity, suggesting an interplay between the two isoforms that may underlie the observed results. Overall, these findings suggest that in PC12 and perhaps other neuroendocrine precursor cells, PKCι influences an early differentiation decision between the neuroendocrine (chromaffin) and sympathetic neuron cell lineages, potentially by affecting PKCζ function.
{"title":"Expression of PKC iota affects neuronal differentiation of PC12 cells at least partly independent of kinase function.","authors":"Alana Doonachar, Alan R Schoenfeld","doi":"10.4236/cellbio.2014.31001","DOIUrl":"https://doi.org/10.4236/cellbio.2014.31001","url":null,"abstract":"<p><p>Atypical PKC (aPKC) plays a role in establishing cell polarity and has been indicated in neuronal differentiation and polarization, including neurite formation in rat pheochromocytoma PC12 cells, albeit by unclear mechanisms. Here, the role of the aPKC isoform, PKC iota (PKCι), in the early neuronal differentiation of PC12 cells was investigated. NGF-treated PC12 cells with stably expressed exogenous wild-type PKCι showed decreased expression of a neuroendocrine marker, increased expression of a neuronal marker, and increased neurite formation. Stable expression of a kinase- inactive PKCι, but not constitutively active PKCι lacking a regulatory domain, had similar although less potent effects. Pharmacological inhibition of endogenous aPKC kinase activity in parental PC12 cells did not inhibit neurite formation, suggesting that some of the observed effects of PKCι expression on neuronal differentiation are kinase- independent. Interestingly, exogenous expression of wild-type and kinase-inactive PKCι had little effect on overall PKCι activity, but caused a decrease in PKC zeta (PKCζ) kinase activity, suggesting an interplay between the two isoforms that may underlie the observed results. Overall, these findings suggest that in PC12 and perhaps other neuroendocrine precursor cells, PKCι influences an early differentiation decision between the neuroendocrine (chromaffin) and sympathetic neuron cell lineages, potentially by affecting PKCζ function.</p>","PeriodicalId":69463,"journal":{"name":"细胞生物学(英文)","volume":"3 1","pages":"1-13"},"PeriodicalIF":0.0,"publicationDate":"2014-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4045628/pdf/nihms573532.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"32408819","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2012-12-18DOI: 10.4236/cellbio.2012.12004
Innocent H Yamodo, Scott D Blystone
The Calcium Integrin Binding protein (CIB) has been identified as interacting specifically with the cytoplasmic tail of the integrin αIIb domain to induce receptor activation and integrin αIIbβ3 mediated cell adhesion to extracellular proteins. In K562 cells stably expressing mutated integrin αVβ3, or chimeric αVβ3 carrying αIIb cytoplasmic tail, we report that the interaction of CIB with β3 integrins is not αIIbβ3 specific but binds αIIb as well as αV cytoplasmic tail domains. A double mutation of two proline residues to alanine residues in the αIIb cytoplasmic domain, previously shown to disturb its conformation, inhibits chimeric αV/αIIbβ3-CIB interaction. This demonstrates that αIIb cytoplasmic domain loop-like conformation is required for interaction with CIB. Moreover, mutations of β3 cytoplasmic domain residues Tyr-747 and/or Tyr-759 to phenylalanine residues (Y747F, Y759F, and Y747,759F) as well as residues Ser-752 to proline or alanine (S752P and S752A), do not affect the αIIbβ3 or αVβ3 interaction with CIB. Since tyrosine residues Tyr-747 and/or Tyr-759 are the sites of tyrosine phosphorylation of β3 subunit, these results suggest that the β3 integrin-CIB interaction occurs through a β3-phosphorylation independent mechanism. Likewise, ablation of conformation-dependent affinity change in β3 Ser752Pro mutation had no affect on CIB-β3 interaction. In summary, our results demonstrate that the αIIb-subunit integrin and CIB interaction is non-exclusive and requires the loop-like αIIb-cytoplasmic domain conformation. An interaction of CIB with αV-containing integrins provides an additional role for this molecule in keeping with its expression outside of platelets.
{"title":"Calcium Integrin Binding Protein Associates with Integrins α<sub>V</sub>β<sub>3</sub> and α<sub>IIb</sub>β<sub>3</sub> Independent of β<sub>3</sub> Activation Motifs.","authors":"Innocent H Yamodo, Scott D Blystone","doi":"10.4236/cellbio.2012.12004","DOIUrl":"https://doi.org/10.4236/cellbio.2012.12004","url":null,"abstract":"<p><p>The Calcium Integrin Binding protein (CIB) has been identified as interacting specifically with the cytoplasmic tail of the integrin α<sub>IIb</sub> domain to induce receptor activation and integrin α<sub>IIb</sub>β<sub>3</sub> mediated cell adhesion to extracellular proteins. In K562 cells stably expressing mutated integrin α<sub>V</sub>β<sub>3</sub>, or chimeric α<sub>V</sub>β<sub>3</sub> carrying α<sub>IIb</sub> cytoplasmic tail, we report that the interaction of CIB with β<sub>3</sub> integrins is not α<sub>IIb</sub>β<sub>3</sub> specific but binds α<sub>IIb</sub> as well as α<sub>V</sub> cytoplasmic tail domains. A double mutation of two proline residues to alanine residues in the α<sub>IIb</sub> cytoplasmic domain, previously shown to disturb its conformation, inhibits chimeric α<sub>V</sub>/α<sub>IIb</sub>β<sub>3</sub>-CIB interaction. This demonstrates that α<sub>IIb</sub> cytoplasmic domain loop-like conformation is required for interaction with CIB. Moreover, mutations of β<sub>3</sub> cytoplasmic domain residues Tyr-747 and/or Tyr-759 to phenylalanine residues (Y747F, Y759F, and Y747,759F) as well as residues Ser-752 to proline or alanine (S752P and S752A), do not affect the α<sub>IIb</sub>β<sub>3</sub> or α<sub>V</sub>β<sub>3</sub> interaction with CIB. Since tyrosine residues Tyr-747 and/or Tyr-759 are the sites of tyrosine phosphorylation of β<sub>3</sub> subunit, these results suggest that the β<sub>3</sub> integrin-CIB interaction occurs through a β<sub>3</sub>-phosphorylation independent mechanism. Likewise, ablation of conformation-dependent affinity change in β<sub>3</sub> Ser752Pro mutation had no affect on CIB-β<sub>3</sub> interaction. In summary, our results demonstrate that the α<sub>IIb</sub>-subunit integrin and CIB interaction is non-exclusive and requires the loop-like α<sub>IIb</sub>-cytoplasmic domain conformation. An interaction of CIB with α<sub>V</sub>-containing integrins provides an additional role for this molecule in keeping with its expression outside of platelets.</p>","PeriodicalId":69463,"journal":{"name":"细胞生物学(英文)","volume":"1 2","pages":"30-37"},"PeriodicalIF":0.0,"publicationDate":"2012-12-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3807131/pdf/nihms433379.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40269480","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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