Analysis of Small GTPase Signaling Pathways Using p21-activated Kinase Mutants That Selectively Couple to Cdc42

Reeder, Melissa; Serebriiskii, Ilya G.; Golemis, Erica A.; Chernoff, Jonathan

doi:10.1074/jbc.m103925200

Cited by 32 publications

(26 citation statements)

References 50 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Interestingly, the mutation of the residue Phe 91 of PAK3, homologous to the Phe 96 of PAK1, abolished Rac binding more strongly than Cdc42 binding (33). Consistent with this, the binding of the PAK3b isoform is more impeded for Rac than for Cdc42.…”

Section: Discussionsupporting

confidence: 65%

“…Moreover, this region of GTPase binding that encompasses the CRIB domain is also implicated in the selectivity of the interaction with Rac versus Cdc42 (33,49). Interestingly, the mutation of the residue Phe 91 of PAK3, homologous to the Phe 96 of PAK1, abolished Rac binding more strongly than Cdc42 binding (33).…”

Section: Discussionmentioning

confidence: 99%

“…Co-immunoprecipitation was performed as described by Reeder et al (33). Briefly, COS-7 cells were transfected with 5 g of active GFPtagged Rac or Cdc42 along with 5 g of HA-tagged PAK3a and PAK3b constructs.…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

A New Constitutively Active Brain PAK3 Isoform Displays Modified Specificities toward Rac and Cdc42 GTPases

Rousseau¹,

Goupille²,

Morin³

et al. 2003

Journal of Biological Chemistry

View full text Add to dashboard Cite

p21-activated kinases (PAK) are involved in the control of cytoskeleton dynamics and cell cycle progression. Here we report the characterization of a new mammalian PAK3 mRNA that contains a 45-bp alternatively spliced exon. This exon encodes for 15 amino acids that are inserted in the regulatory domain, inside the autoinhibitory domain but outside the Cdc42 and Rac interactive binding domain. The transcript of the 68-kDa new isoform named PAK3b is expressed in various areas of the adult mouse brain. In contrast to PAK3 without the exon b (PAK3a), whose basal kinase activity is weak in resting cells, PAK3b displays a high kinase activity in starved cells that is not further stimulated by active GTPases. Indeed, we demonstrate that the autoinhibitory domain of PAK3b no longer inhibits the kinase activity of PAK3. Moreover, we show that the 15-amino acid insertion within the autoinhibitory domain impedes the ability of PAK3b to bind to the GTPases Rac and Cdc42 and changes its specificity toward the GTPases. Altogether, our results show that the new PAK3b isoform has unique properties and would signal differently from PAK3a in neurons.

show abstract

Section: Discussionsupporting

confidence: 65%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

A New Constitutively Active Brain PAK3 Isoform Displays Modified Specificities toward Rac and Cdc42 GTPases

Rousseau¹,

Goupille²,

Morin³

et al. 2003

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…In order to test for Cdc42 activation by CB2 SH3+ and CB2 SH3-, we used a well-characterized assay system that relies on 2790 Journal of Cell Science 124 (16) the interaction between activated Cdc42 and p21-activated kinase 1 (Pak1). Pak1 is a downstream effector of activated Cdc42 and interacts with GTP-bound Cdc42 (Reeder et al, 2001). The minimum sequence required for binding has been mapped and is routinely used to assay for levels of activated Cdc42 inside cells.…”

Section: Cb2 Sh3- But Not Cb2 Sh3+ Forms a Ternary Complex With Gementioning

confidence: 99%

Collybistin splice variants differentially interact with gephyrin and Cdc42 to regulate gephyrin clustering at GABAergic synapses

Tyagarajan

Ghosh

Harvey

et al. 2011

Journal of Cell Science

View full text Add to dashboard Cite

SummaryCollybistin (CB) is a guanine-nucleotide-exchange factor (GEF) selectively activating Cdc42. CB mutations cause X-linked mental retardation due to defective clustering of gephyrin, a postsynaptic protein associated with both glycine and GABA A receptors. Using a combination of biochemistry and cell biology we provide novel insights into the roles of the CB2 splice variants, CB2 SH3+ and CB2 SH3-, and their substrate, Cdc42, in regulating gephyrin clustering at GABAergic synapses. Transfection of Myc-tagged CB2 SH3+ and CB2 SH3-into cultured neurons revealed strong, but distinct, effects promoting postsynaptic gephyrin clustering, denoting mechanistic differences in their function. In addition, overexpression of constitutively active or dominant-negative Cdc42 mutants identified a new function of Cdc42 in regulating the shape and size of postsynaptic gephyrin clusters. Using biochemical assays and native brain tissue, we identify a direct interaction between gephyrin and Cdc42, independent of its activation state. Finally, our data show that CB2 SH3-, but not CB2 SH3+ , can form a ternary complex with gephyrin and Cdc42, providing a biochemical substrate for the distinct contribution of these CB isoforms in gephyrin clustering at GABAergic postsynaptic sites. Taken together, our results identify CB and Cdc42 as major regulators of GABAergic postsynaptic densities.

show abstract

“…These findings suggest that the CRIB-mediated interaction with activated Rac1/Cdc42 is generally required for Pak-mediated cell transformation. The AID is involved not only in the inhibition of the kinase domain, but also in the specific recognition of GTPase (Lei et al, 2000;Reeder et al, 2001). Thus, it is likely that the CRIB motif and the AID of MoPak are unmasked because of the unique conformational features of the AID, thereby allowing efficient access to activated Rac1/Cdc42 (Figure 7d).…”

Section: Discussionmentioning

confidence: 99%

Functional transition of Pak proto-oncogene during early evolution of metazoans

et al. 2010

View full text Add to dashboard Cite

Proto-oncogenes encode signaling molecular switches regulating cellular homeostasis in metazoans, and can be converted to oncogenes by gain-of-function mutations. To address the molecular basis for development of the regulatory system of proto-oncogenes during evolution, we screened for ancestral proto-oncogenes from the unicellular choanoflagellate Monosiga ovata by monitoring their transforming activities, and isolated a Pak gene ortholog encoding a serine/threonine kinase as a 'primitive oncogene'. We also cloned Pak orthologs from fungi and the multicellular sponge Ephydatia fluviatilis, and compared their regulatory features with that of M. ovata Pak (MoPak). MoPak is constitutively active and induces cell transformation in mammalian fibroblasts, although the Pak orthologs from multicellular animals are strictly regulated. Analyses of Pak mutants revealed that structural alteration of the auto-inhibitory domain (AID) of MoPak confers higher constitutive kinase activity, as well as greater binding ability to Rho family GTPases than the multicellular Paks, and this structural alteration is responsible for cell transformation and disruption of multicellular tissue organization. These results show that maturation of AID function was required for the development of the strict regulatory system of the Pak proto-oncogene, and suggest a potential link between the establishment of the regulatory system of proto-oncogenes and metazoan evolution.

show abstract

Analysis of Small GTPase Signaling Pathways Using p21-activated Kinase Mutants That Selectively Couple to Cdc42

Cited by 32 publications

References 50 publications

A New Constitutively Active Brain PAK3 Isoform Displays Modified Specificities toward Rac and Cdc42 GTPases

A New Constitutively Active Brain PAK3 Isoform Displays Modified Specificities toward Rac and Cdc42 GTPases

Collybistin splice variants differentially interact with gephyrin and Cdc42 to regulate gephyrin clustering at GABAergic synapses

Functional transition of Pak proto-oncogene during early evolution of metazoans

Contact Info

Product

Resources

About