Molecular Dissection of the Interaction between the Small G Proteins Rac1 and RhoA and Protein Kinase C-related Kinase 1 (PRK1)

Owen, Darerca; Lowe, Peter N.; Nietlispach, Daniel; Brosnan, C. Elaine; Chirgadze, Dimitri Y.; Parker, Peter J.; Blundell, Tom L.; Mott, Helen R.

doi:10.1074/jbc.m304313200

Cited by 50 publications

(88 citation statements)

References 65 publications

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“…The K d for full-length BART, BART-(1-136), and BART-(14 -136) were 40, 41, and 34 nM, respectively, indicating that neither the extreme N terminus nor the C terminus is required for the interaction with Arl2. These affinities are in agreement with the previously published K d of the interaction (41,42) and is similar to the affinities we have observed for other small G protein-effector complexes (25,43).…”

Section: Figure 2 Packing Interactions That Give Rise To the Unusualsupporting

confidence: 82%

The Structure of Binder of Arl2 (BART) Reveals a Novel G Protein Binding Domain

Bailey¹,

Campbell

Evetts

et al. 2009

Journal of Biological Chemistry

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The ADP-ribosylation factor-like (Arl) family of small G proteins are involved in the regulation of diverse cellular processes. Arl2 does not appear to be membrane localized and has been implicated as a regulator of microtubule dynamics. The downstream effector for Arl2, Binder of Arl 2 (BART) has no known function but, together with Arl2, can enter mitochondria and bind the adenine nucleotide transporter. We have solved the solution structure of BART and show that it forms a novel fold composed of six ␣-helices that form three interlocking "L" shapes. Analysis of the backbone dynamics reveals that the protein is highly anisotropic and that the loops between the central helices are dynamic. The regions involved in the binding of Arl2 were mapped onto the surface of BART and are found to localize to these loop regions. BART has faces of differing charge and structural elements, which may explain how it can interact with other proteins.

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Section: Figure 2 Packing Interactions That Give Rise To the Unusualsupporting

confidence: 82%

The Structure of Binder of Arl2 (BART) Reveals a Novel G Protein Binding Domain

Bailey¹,

Campbell

Evetts

et al. 2009

Journal of Biological Chemistry

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“…An alanine-to-lysine mutation was introduced into both the HR1a and HR1b GTPase-binding domains of mouse PRK2 to generate mPRK2(A66K,A155K). Based on structural studies of the related protein PRK1, these point mutations are predicted to prevent GTPase binding (27). The results of coimmunoprecipitation experiments confirmed that wild-type mPRK2 interacts with a constitutively activated version of RhoA (L63RhoA) but mPRK2(A66K,A155K) does not (Fig.…”

Section: Resultssupporting

confidence: 50%

The Rho Target PRK2 Regulates Apical Junction Formation in Human Bronchial Epithelial Cells

Wallace

Magalhaes

Hall

2011

Molecular and Cellular Biology

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Rho GTPases regulate multiple signaling pathways to control a number of cellular processes during epithelial morphogenesis. To investigate the downstream pathways through which Rho regulates epithelial apical junction formation, we screened a small interfering RNA (siRNA) library targeting 28 known Rho target proteins in 16HBE human bronchial epithelial cells. This led to the identification of the serine-threonine kinase PRK2 (protein kinase C-related kinase 2, also called PKN2). Depletion of PRK2 does not block the initial formation of primordial junctions at nascent cell-cell contacts but does prevent their maturation into apical junctions. PRK2 is recruited to primordial junctions, and this localization depends on its C2-like domain. Rho binding is essential for PRK2 function and also facilitates PRK2 recruitment to junctions. Kinase-dead PRK2 acts as a dominant-negative mutant and prevents apical junction formation. We conclude that PRK2 is recruited to nascent cell-cell contacts through its C2-like and Rho-binding domains and promotes junctional maturation through a kinase-dependent pathway.

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“…Previous work also found that the affinity of Cdc42 for IQGAP1 was substantially higher than that for the CRIB effectors, PAK1 and WASP (12); however, these data again were derived from an indirect assay (inhibition of GTP hydrolysis), which may explain deviation from data presented here. We believe that the direct analysis we use (36,47,52,60) and present here provides the most accurate estimation to date of the relative binding affinities.…”

Section: Discussionmentioning

confidence: 99%

“…In our previous work on ACK (a Cdc42 effector) and PRK1 (a Rac1 effector), we designed a number of Cdc42 and Rac1 mutants that abrogated or significantly reduced binding to these effectors, along with others that were in or near the binding interface but were not energetically involved with those particular interactions (47,52). We therefore examined the binding of IQGAP1 to those mutants of Cdc42 and Rac1 that were designed to lie in the interface with ACK or PRK1, respectively, and did not fall into any of the previous three categories of mutants (Table 4), to determine whether Cdc42 or Rac1 utilizes a similar binding surface to interact with IQGAP1 and the other effectors.…”

Section: Mutations In the Insert Loop-thementioning

confidence: 99%

The IQGAP1-Rac1 and IQGAP1-Cdc42 Interactions

Owen

Campbell

Littlefield

et al. 2008

Journal of Biological Chemistry

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IQGAP1 contains a domain related to the catalytic portion of the GTPase-activating proteins (GAPs) for the Ras small G proteins, yet it has no RasGAP activity and binds to the Rho family small G proteins Cdc42 and Rac1. It is thought that IQGAP1 is an effector of Rac1 and Cdc42, regulating cell-cell adhesion through the E-cadherin-catenin complex, which controls formation and maintenance of adherens junctions. This study investigates the binding interfaces of the Rac1-IQGAP1 and Cdc42-IQGAP1 complexes. We mutated Rac1 and Cdc42 and measured the effects of mutations on their affinity for IQGAP1. We have identified similarities and differences in the relative importance of residues used by Rac1 and Cdc42 to bind IQGAP1. Furthermore, the residues involved in the complexes formed with IQGAP1 differ from those formed with other effector proteins and GAPs. Relatively few mutations in switch I of Cdc42 or Rac1 affect IQGAP1 binding; only mutations in residues 32 and 36 significantly decrease affinity for IQGAP1. Switch II mutations also affect binding to IQGAP1 although the effects differ between Rac1 and Cdc42; mutation of either Asp-63, Arg-68, or Leu-70 abrogate Rac1 binding, whereas no switch II mutations affect Cdc42 binding to IQGAP1. The Rho family "insert loop" does not contribute to the binding affinity of Rac1/ Cdc42 for IQGAP1. We also present thermodynamic data pertaining to the Rac1/Cdc42-RhoGAP complexes. Switch II contributes a large portion of the total binding energy to these complexes, whereas switch I mutations also affect binding. In addition we identify "cold spots" in the Rac1/Cdc42-RhoGAP/ IQGAP1 interfaces. Competition data reveal that the binding sites for IQGAP1 and RhoGAP on the small G proteins overlap only partially. Overall, the data presented here suggest that, despite their 71% identity, Cdc42 and Rac1 appear to have only partially overlapping binding sites on IQGAP1, and each uses different determinants to achieve high affinity binding.The Rho family of small G proteins are well documented to regulate cell motility through their regulation of the actin cytoskeleton (1). These small G proteins act as molecular switches, being active in the GTP-bound form and inactive in the GDP-bound form. The interaction of the active, GTPbound, G protein with an effector protein triggers a downstream signaling cascade. Rho family proteins, acting via these effector proteins, have been shown to control the polymerization of actin in the peripheral cortex of the cell. Cdc42 controls filopodia formation; these are actin-rich fingers that establish the direction of motility. Rac1 controls lamellipodia formation; these actin-rich ruffles at the leading edge of the cell initiate motion, whereas Rho controls the establishment of stress fibers whose formation results in the contractile force that moves the body of the cell behind the leading edge (2).Effector proteins for two members of the Rho family, Cdc42 and Rac1, include the PAK 3 family of serine/threonine kinases reviewed in Ref. 3, the ACK tyrosine kin...

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Molecular Dissection of the Interaction between the Small G Proteins Rac1 and RhoA and Protein Kinase C-related Kinase 1 (PRK1)

Cited by 50 publications

References 65 publications

The Structure of Binder of Arl2 (BART) Reveals a Novel G Protein Binding Domain

The Structure of Binder of Arl2 (BART) Reveals a Novel G Protein Binding Domain

The Rho Target PRK2 Regulates Apical Junction Formation in Human Bronchial Epithelial Cells

The IQGAP1-Rac1 and IQGAP1-Cdc42 Interactions

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