Identification of the Phosphatidylserine Binding Site in the C2 Domain that Is Important for PKC Activation and in Vivo Cell Localiz
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- 作者：Pablo Conesa-Zamora ; Maria Jose Lopez-Andreo ; Juan C. Gó ; mez-Ferná ; ndez ; and Senena Corbalá ; n-Garcí ; a
- 刊名：Biochemistry
- 出版年：2001
- 出版时间：November 20, 2001
- 年：2001
- 卷：40
- 期：46
- 页码：13898 - 13905
- 全文大小：146K
- 年卷期：v.40,no.46(November 20, 2001)
- ISSN：1520-4995
文摘
The C2 domain of classical PKCs binds to membranes through Ca2+ bridging to phosphatidylserine as recently observed through X-ray diffraction of the isolated domain. Additionally, it has beenproposed that N189, T251, R216, and R249A interact directly with phosphatidylserine [Verdaguer, N., etal. (1999) EMBO J. 18, 6329-6338]. When these four residues were mutated to Ala to determine theirrole in PKC binding to phospholipid membranes, PKC activation, and in its in vivo localization, theresults revealed that they were very important for the activation of full-length PKC
. N189, in particular,was involved in the activation of the enzyme after its interaction with PS, since its mutation to Ala didnot decrease the level of membrane binding but did prevent full enzyme activation. On the other hand,mutations R216A, R249A, and T251A affected both membrane binding and enzyme activation, althoughT251A had the most drastic effect, suggesting that the protein interactions with the carbonyl groups ofthe phospholipid are also a key event in the activation process. Taken together, these results show that thefour residues located near the calcium binding site are critical in phosphatidylserine-dependent PKCactivation, in which N189 plays an important role, triggering the enzyme activation probably by interactingwith neighboring residues of the protein when lipid binding occurs. Furthermore, these results providestrong evidence for better defining one of the two phosphatidylserine isomer models proposed in theprevious crystallographic report.
. N189, in particular,was involved in the activation of the enzyme after its interaction with PS, since its mutation to Ala didnot decrease the level of membrane binding but did prevent full enzyme activation. On the other hand,mutations R216A, R249A, and T251A affected both membrane binding and enzyme activation, althoughT251A had the most drastic effect, suggesting that the protein interactions with the carbonyl groups ofthe phospholipid are also a key event in the activation process. Taken together, these results show that thefour residues located near the calcium binding site are critical in phosphatidylserine-dependent PKCactivation, in which N189 plays an important role, triggering the enzyme activation probably by interactingwith neighboring residues of the protein when lipid binding occurs. Furthermore, these results providestrong evidence for better defining one of the two phosphatidylserine isomer models proposed in theprevious crystallographic report.