ABCMdb

ABCC8 p.Ala365Glu

Predicted by SNAP2:	C: N (93%), D: N (66%), E: N (61%), F: N (61%), G: N (87%), H: N (72%), I: N (87%), K: N (61%), L: N (82%), M: N (66%), N: N (78%), P: N (57%), Q: N (82%), R: N (66%), S: N (97%), T: N (97%), V: N (93%), W: D (75%), Y: N (66%),
Predicted by PROVEAN:	C: N, D: D, E: D, F: D, G: N, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: N, R: D, S: N, T: N, V: N, W: D, Y: D,

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[hide] The p21-activated kinase 3 implicated in mental re... J Biol Chem. 2007 Jul 20;282(29):21497-506. Epub 2007 May 30. Kreis P, Thevenot E, Rousseau V, Boda B, Muller D, Barnier JV
The p21-activated kinase 3 implicated in mental retardation regulates spine morphogenesis through a Cdc42-dependent pathway.
J Biol Chem. 2007 Jul 20;282(29):21497-506. Epub 2007 May 30., [PMID:17537723]

Abstract [show]
The p21-activated kinase 3 (PAK3) is one of the recently identified genes for which mutations lead to nonsyndromic mental retardation. PAK3 is implicated in dendritic spine morphogenesis and is a key regulator of synaptic functions. However, the underlying roles of PAK3 in these processes remain poorly understood. We report here that the three mutations R419X, A365E, and R67C, responsible for mental retardation have different effects on the biological functions of PAK3. The R419X and A365E mutations completely abrogate the kinase activity. The R67C mutation drastically decreases the binding of PAK3 to the small GTPase Cdc42 and impairs its subsequent activation by this GTPase. We also report that PAK3 binds significantly more Cdc42 than Rac1 and is selectively activated by endogenous Cdc42, suggesting that PAK3 is a specific effector of Cdc42. Interestingly, the expression of the three mutated proteins in hippocampal neurons affects spinogenesis differentially. Both kinase-dead mutants slightly decrease the number of spines but profoundly alter spine morphology, whereas expression of the R67C mutant drastically decreases spine density. These results demonstrate that the Cdc42/PAK3 is a key module in dendritic spine formation and synaptic plasticity.

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No. Sentence Comment
3 We report here that the three mutations R419X, A365E, and R67C, responsible for mental retardation have different effects on the biological functions of PAK3. Login to comment
4 The R419X and A365E mutations completely abrogate the kinase activity. Login to comment
22 Two mutations, R419X and A365E, are located in the kinase domain, whereas the third one, R67C, is present in the regulatory domain, at the N-terminal end of the p21-GTPase-binding domain (PBD). Login to comment
23 We show that the two mutations R419X and A365E completely inactivate the catalytic function of the kinase. Login to comment
42 The mutations introduced into PAK3 include the three mutations responsible for mental retardation: R67C (oligonucleotide set: 5Ј-ccaataagaagaaagagaaagagtgcccagagatctct- cttcc-3Ј and 5Ј-ggaagagagatctctgggcactctttctctttcttcttattgg-3Ј) leading to the HA-PAK3a-R67C plasmid; A365E (oligonucleotide set: 5Ј-gtatggatgaaggacagatagaagctgtctgtagagagtgcc-3Ј and 5Ј-ggcactctctacagacagcttctatctgtccttcatccatac-3Ј) leading to the HA-PAK3a-A365E plasmid; R419X (oligonucleotide set: 5Ј-cactcctgagcaaagtaaatgaagcactatggtgggaac-3Ј and 5Ј-gttccc- accatagtgcttcatttactttgctcaggagtg-3Ј) leading to the HA-PAK3-R419X plasmid. Login to comment
45 The BamHI/XbaI fragments of the different HA-tagged PAK3 constructs described above were subcloned into the BamHI/XbaI-linearized pEGFP vector (Clontech, Ozyme, St. Quentin en Yvelines, France), to obtain GFP-tagged PAK3 plasmids named GFP-PAK3-wt, GFP-PAK3-R67C, GFP-PAK3-A365E, GFP-PAK3-R419X, GFP-PAK3-H78L-H81L, and GFP-PAK3-K297L. Login to comment
95 The second mutation, in the kinase domain, corresponds to a substitution of a conserved alanine residue to a glutamate (A365E) (9). Login to comment
97 The molecular defects associated with the A365E and R67C mutations have not yet beendescribed.ToassesstheeffectsofthethreemutationsR419X, A365E, and R67C, on the biochemical properties of PAK3, we introduced them into the coding sequence of the PAK3 gene. Login to comment
103 Arrows indicate the localization of the three mutations R67C, A365E, and R419X, responsible for mental retardation, whereas arrowheads indicate mutations described in the literature: the H78L-H81L mutation located in the PBD disrupts the binding to GTPases, and the K297L mutation disorganizes the ATP binding pocket in the catalytic domain. Login to comment
106 B, R419X and A365E mutations abolish kinase activity, whereas R67C mutation has no effect on the kinase activity. Login to comment
107 PAK3 constructs: wt, K297L kinase-dead mutant, and the three mental retardation mutants (R419X, A365E, and R67C) were co-transfected in COS7 cells with active V12 or inactive N17 mutants of GFP-Cdc42 constructs. Login to comment
116 We also report here that the A365E mutation completely abolished the kinase activity of PAK3. Login to comment
120 Thus, contrary to the R419X and A365E mutants, which are devoid of kinase activity, the kinase activity of the R67C mutant is similar to that of the wild-type PAK3 protein, in these experimental conditions. Login to comment
139 We also analyzed by co-immunoprecipitation the interaction of Cdc42 with the three kinase-dead mutants PAK3-K297L, PAK3-R419X, and PAK3-A365E. Login to comment
197 The three kinase-dead proteins K297L, R419X, and A365E and the two mutants affecting GTPase binding, H78L-H81L and R67C, displayed the same subcellular localization as the PAK3-wt protein. Login to comment
201 For this, R419X, A365E, and R67C mutants were co-expressed with EGFP in pyramidal cells through biolistic transfection. Login to comment
219 Interestingly, expression of the A365E mutant (Fig. 6C) produced a phenotype comparable to the R419X mutant, although with a slightly different balance between the two types of alterations. Login to comment
220 As illustrated in Fig. 6E, the reduction in protrusion density was more marked in most A365E-transfected cells than had been observed with the R419X mutation (0.69 Ϯ 0.07 protrusions/␮m; n ϭ 8; p Ͻ 0.05), whereas the increase in elongated immature protrusions was comparable, as indicated by the shift observed in the distribution of protrusion length (Fig. 6F; mean length: 1.50 Ϯ 0.07 versus 1.14 Ϯ 0.03 ␮m, n ϭ 8-12; p Ͻ 0.05). Login to comment
227 The two mutations R419X and A365E, located in the catalytic domain inactivate the kinase activity. Login to comment
228 This result was previously reported for the R419X-truncated protein, but was unpredicted for the A365E mutant (7, 9). Login to comment
229 This A365E mutation is located in a conserved subdomain named VIa inside the ␣ helix E of the large lobe of the kinases (31). Login to comment
238 GFP-PAK3 constructs: wild-type, K297L kinase- deadmutant,H78L-H81LGTPase-bindingmutant,andthethreementalretarda- tion mutants R419X, A365E, and R67C (left panel, green fluorescence) were co- transfectedinhippocampalneuronswithamRFP-actinplasmid(redinthemiddle panel) at DIV 18 and fixed at DIV 21. Login to comment
242 A-D, spine morphology obtained from neurons co-transfected with EGFP and either an empty vector (A) or PAK3-R419X (B), PAK3-A365E (C), and PAK3-R67C (D); scale bar: 2 ␮m. E, decrease in protrusion density in pyramidal neurons transfected with the three PAK3 mutants. Login to comment
244 F, formation of elongated, immature protrusions analyzed by measuring the protrusion length in cells transfected with an EGFP vector (Ctrl) or PAK3 mutants (R419X, A365E, and R67C). Login to comment
274 We report here that expression of the kinase-dead PAK3-A365E mutant induced a similar phenotype, whereas expression of PAK3-R67C, defective in Cdc42 binding and in activation by Cdc42, led to a pronounced decrease of spine density. Login to comment
275 Altogether these results show that both inactivation of the kinase activity (R419X and A365E) and defect of binding and activation by Cdc42 (R67C) led to spine anomalies, suggesting that PAK3 binding to Cdc42 and PAK3 kinase activity are both necessary for spinogenesis. Login to comment

[hide] p21-Activated kinase 3 (PAK3) protein regulates sy... J Biol Chem. 2011 Nov 18;286(46):40044-59. Epub 2011 Sep 23. Thevenot E, Moreau AW, Rousseau V, Combeau G, Domenichini F, Jacquet C, Goupille O, Amar M, Kreis P, Fossier P, Barnier JV
p21-Activated kinase 3 (PAK3) protein regulates synaptic transmission through its interaction with the Nck2/Grb4 protein adaptor.
J Biol Chem. 2011 Nov 18;286(46):40044-59. Epub 2011 Sep 23., [PMID:21949127]

Abstract [show]
Mutations in the p21-activated kinase 3 gene (pak3) are responsible for nonsyndromic forms of mental retardation. Expression of mutated PAK3 proteins in hippocampal neurons induces abnormal dendritic spine morphology and long term potentiation anomalies, whereas pak3 gene invalidation leads to cognitive impairments. How PAK3 regulates synaptic plasticity is still largely unknown. To better understand how PAK3 affects neuronal synaptic plasticity, we focused on its interaction with the Nck adaptors that play a crucial role in PAK signaling. We report here that PAK3 interacts preferentially with Nck2/Grb4 in brain extracts and in transfected cells. This interaction is independent of PAK3 kinase activity. Selective uncoupling of the Nck2 interactions in acute cortical slices using an interfering peptide leads to a rapid increase in evoked transmission to pyramidal neurons. The P12A mutation in the PAK3 protein strongly decreases the interaction with Nck2 but only slightly with Nck1. In transfected hippocampal cultures, expression of the P12A-mutated protein has no effect on spine morphogenesis or synaptic density. The PAK3-P12A mutant does not affect synaptic transmission, whereas the expression of the wild-type PAK3 protein decreases the amplitude of spontaneous miniature excitatory currents. Altogether, these data show that PAK3 down-regulates synaptic transmission through its interaction with Nck2.

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No. Sentence Comment
48 The following plasmids were described previously (10, 24): the pcDNA3-HA-PAK3-WT, -kd, -ca, -R419X, -A365E, and -R67C plasmids encode mouse HA-tagged PAK3 wild-type, K297L kinase-defective, T421E constitutively active, mental retardation truncated mutant, missense kinase defective mutant, and the missense R67C mutated proteins, respectively. Login to comment
183 We also tested the capacity of the mental retardation mutated A365E and R419X PAK3 proteins to interact with Nck2 in co-immunoprecipitation assays, and no difference was observed between these mutated proteins compared with the wild type (Fig. 2D). Login to comment
231 D, three PAK3 proteins bearing the mental retardation mutation A365E, R419Stop, and R67C co-immunoprecipitate with Nck2 with an apparent normal efficiency. Login to comment