ABCMdb

PMID: 17537723

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., [PubMed]

Sentences

No. Mutations Sentence Comment

3 ABCC8 p.Ala365Glu 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 ABCC8 p.Ala365Glu The R419X and A365E mutations completely abrogate the kinase activity. Login to comment 22 ABCC8 p.Ala365Glu 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 ABCC8 p.Ala365Glu We show that the two mutations R419X and A365E completely inactivate the catalytic function of the kinase. Login to comment 42 ABCC8 p.Ala365Glu ABCC8 p.Ala365Glu 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 ABCC8 p.Ala365Glu 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 ABCC8 p.Ala365Glu 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 ABCC8 p.Ala365Glu ABCC8 p.Ala365Glu 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 ABCC8 p.Ala365Glu 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 ABCC8 p.Ala365Glu B, R419X and A365E mutations abolish kinase activity, whereas R67C mutation has no effect on the kinase activity. Login to comment 107 ABCC8 p.Ala365Glu 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 ABCC8 p.Ala365Glu We also report here that the A365E mutation completely abolished the kinase activity of PAK3. Login to comment 120 ABCC8 p.Ala365Glu 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 ABCC8 p.Ala365Glu 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 ABCC8 p.Ala365Glu 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 ABCC8 p.Ala365Glu For this, R419X, A365E, and R67C mutants were co-expressed with EGFP in pyramidal cells through biolistic transfection. Login to comment 219 ABCC8 p.Ala365Glu 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 ABCC8 p.Ala365Glu 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 ABCC8 p.Ala365Glu The two mutations R419X and A365E, located in the catalytic domain inactivate the kinase activity. Login to comment 228 ABCC8 p.Ala365Glu This result was previously reported for the R419X-truncated protein, but was unpredicted for the A365E mutant (7, 9). Login to comment 229 ABCC8 p.Ala365Glu 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 ABCC8 p.Ala365Glu 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 ABCC8 p.Ala365Glu 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 ABCC8 p.Ala365Glu 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 ABCC8 p.Ala365Glu 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 ABCC8 p.Ala365Glu 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