Structure of the Complex of Cdc42Hs with a Peptide Derived from P-21 Activated Kinase<sup>,</sup>

Gizachew, Dawit; Guo, Wei; Chohan, Kamaldeep K.; Sutcliffe, Michael J.; Oswald, Robert E.

doi:10.1021/bi992646d

Cited by 40 publications

(69 citation statements)

References 61 publications

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“…6A and B). The elution profile of the RD-Cdc42 complex also suggests an apparent molecular mass higher than expected for a monomer, not in line with the proposed activation model and with NMR data showing smaller CRIB fragments in complex with Cdc42 to be monomers (12,27). Since the fragments used for the NMR studies were rather small, they might not contain all of the motifs necessary for dimerization.…”

Section: Resultsmentioning

confidence: 69%

“…The RD-CD complex described here contains all of these regulatory elements plus the PIX (p21-interacting exchange factor) binding motif also involved in the regulation of PAK. The activation model deduced from the crystal and nuclear magnetic resonance (NMR) structures (12,20,27) would predict that the regulatory fragment unfolds and forms a monomer due to the interaction with Cdc42 and that its inhibitory interaction with the CD is released. Using size exclusion chromatography, we find that the regulatory fragment (residues 57 to 200) elutes with an apparent higher molecular mass, arguing that the fragment may still form a possibly rapidly interconverting dimer via the Di motif ( Fig.…”

Section: Resultsmentioning

confidence: 99%

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Conformational Switch and Role of Phosphorylation in PAK Activation

Buchwald

Hostinová

Rudolph

et al. 2001

Molecular and Cellular Biology

101

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p21-activated protein kinases (PAKs) are involved in signal transduction processes initiating a variety of biological responses. They become activated by interaction with Rho-type small GTP-binding proteins Rac and Cdc42 in the GTP-bound conformation, thereby relieving the inhibition of the regulatory domain (RD) on the catalytic domain (CD). Here we report on the mechanism of activation and show that proteolytic digestion of PAK produces a heterodimeric RD-CD complex consisting of a regulatory fragment (residues 57 to 200) and a catalytic fragment (residues 201 to 491), which is active in the absence of Cdc42. Cdc42-GppNHp binds with low affinity (K d 0.6 M) to intact kinase, whereas the affinity to the isolated regulatory fragment is much higher (K d 18 nM), suggesting that the difference in binding energy is used for the conformational change leading to activation. The full-length kinase, the isolated RD, and surprisingly also their complexes with Cdc42 behave as dimers on a gel filtration column. Cdc42-GppNHp interaction with the RD-CD complex is also of low affinity and does not dissociate the RD from the CD. After autophosphorylation of the kinase domain, Cdc42 binds with high (14 nM) affinity and dissociates the RD-CD complex. Assuming that the RD-CD complex mimics the interaction in native PAK, this indicates that the small G protein may not simply release the RD from the CD. It acts in a more subtle allosteric control mechanism to induce autophosphorylation, which in turn induces the release of the RD and thus full activation.GTP-binding proteins of the Ras superfamily are molecular switches which cycle between the GDP-bound off and GTPbound on states. In the on state, they interact with effectors which are defined as molecules interacting more tightly with the GTP-bound form than with the GDP-bound form. By interacting with effectors, they mediate downstream biological effects. The switch returns to the GDP-bound off state by the GTPase reaction which is catalyzed by GTPase-activating proteins (4, 5).Recently, it has become clear that many of the GTP-binding proteins interact with an array of different effectors and thus are involved in more than one signal transduction pathway (26). Many of these effectors are protein kinases that become activated in the course of this interaction. A prominent example are the different Raf kinases (three isoforms) which interact with activated Ras in the course of growth regulation and become activated via a mechanism that involves translocation to the plasma membrane and most likely further allosteric regulatory events which are still incompletely understood.In the case of the Rho subfamily members Rho, Rac, and Cdc42, a number of Ser/Thr-specific protein kinases have been identified, such as protein kinase N and the Rho kinases for Rho. The first kinases to be identified as downstream targets were the Tyr-specific ACK (activated Cdc42-associated kinase) (23) and the Ser/Thr-specific PAK (p21-activated kinase) (24). PAK constitutes a large family of related protein k...

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Section: Resultsmentioning

confidence: 69%

Section: Resultsmentioning

confidence: 99%

Conformational Switch and Role of Phosphorylation in PAK Activation

Buchwald

Hostinová

Rudolph

et al. 2001

Molecular and Cellular Biology

101

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“…The latter findings are consistent with various other lines of evidence from NMR studies. The NMR-derived structures for different activated forms of Cdc42 bound to WASP (Wiscott-Aldrich syndrome protein), Pak, and Ack (activated Cdc42-associated kinase) showed no resonances for either the Switch I or Switch II regions of Cdc42, prior to the addition of the effector protein (16,31,32,35). This was attributed to the dynamic nature of these regions.…”

Section: Discussionmentioning

confidence: 99%

“…This was attributed to the dynamic nature of these regions. However, upon the addition of a Cdc42-effector protein, the resonances for these regions then became clearly defined (16,31,32,35). Additionally, Oswald and co-workers (26) demonstrated in NMR studies that Switch I and Switch II contained significant flexibility and exhibited rapid conformational exchange in both the GDP-and GMP-PCP-bound forms of Cdc42, and that this flexibility was reduced in the Cdc42-GMP-PCP species upon the binding of an effector.…”

Section: Discussionmentioning

confidence: 99%

Effector Proteins Exert an Important Influence on the Signaling-active State of the Small GTPase Cdc42

Phillips

Calero

Chan

et al. 2008

Journal of Biological Chemistry

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GTP-binding (G) proteins regulate the flow of information in cellular signaling pathways by alternating between a GTPbound "active" state and a GDP-bound "inactive" state. Cdc42, a member of the Rho family of Ras-related small G-proteins, plays key roles in the regulation of cell shape, motility, and growth. Here we describe the high resolution x-ray crystal structure for Cdc42 bound to the GTP analog guanylyl ␤,␥-methylenediphosphonate (GMP-PCP) (i.e. the presumed signaling-active state) and show that it is virtually identical to the structures for the signaling-inactive, GDP-bound form of the protein, contrary to what has been reported for Ras and other G-proteins. Especially surprising was that the GMP-PCP-and GDP-bound forms of Cdc42 did not show detectable differences in their Switch I and Switch II loops. Fluorescence studies using a Cdc42 mutant in which a tryptophan residue was introduced at position 32 of Switch I also showed that there was little difference in the Switch I conformation between the GDP-and GMP-PCPbound states (i.e. <10%), which again differed from Ras where much larger changes in Trp-32 fluorescence were observed when comparing these two nucleotide-bound states (>30%). However, the binding of an effector protein induced significant changes in the Trp-32 emission specifically from GMP-PCPbound Cdc42, as well as in the phosphate resonances for GTP bound to this G-protein as indicated in NMR studies. An examination of the available structures for Cdc42 complexed to different effector proteins, versus the x-ray crystal structure for GMP-PCP-bound Cdc42, provides a possible explanation for how effectors can distinguish between the GTP-and GDPbound forms of this G-protein and ensure that the necessary conformational changes for signal propagation occur.Cdc42 is a member of the Rho family of Ras-related small G-proteins and is an essential protein found in all eukaryotic organisms, including yeast, flies, and mammals (1-3). Like Ras, the founding member of the small G-protein family (4), Cdc42 undergoes a GTP-binding/GTP-hydrolytic cycle that enables it to act as a molecular switch in cells. It is activated to undergo GDP-GTP exchange by members of the Dbl family of guanine nucleotide exchange factors (5-7). GTP-bound Cdc42 can bind and/or activate over 20 downstream effector proteins that are responsible for mediating a diversity of cellular functions, including actin cytoskeletal remodeling, cell polarity, intracellular trafficking, epidermal growth factor receptor degradation, and cell cycle progression (1)(2)(3)8). These different signals are terminated when Cdc42 is deactivated through its ability to hydrolyze GTP, a reaction that is catalyzed by GTPase-activating proteins (GAPs) 2 (9). Structural studies of a number of GTP-binding proteins, beginning with the bacterial elongation factor Ef-Tu, and including H-Ras and the ␣ subunits of various members of the family of large G-proteins, have shown that a conserved architecture exists for GTP binding and hydrolytic activity, comprising five...

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“…Residues important for interaction of the PBD region of mammalian PAKs with small GTP-binding proteins were recently identified by nuclear magnetic resonance (37,38) 117 and Met 124 in MIHCK. Possibly these differences account for the fact that the region that includes basic residues N-terminal to the autoregulatory domain of MIHCK increases the affinity of the autoregulatory region to Rac (Fig.…”

Section: Table I Characterization Of the Activity Of Expressed Acanthmentioning

confidence: 99%

Calmodulin-binding and Autoinhibitory Domains ofAcanthamoeba Myosin I Heavy Chain Kinase, a p21-activated Kinase (PAK)

Brzeska

Young

Tan

et al. 2001

Journal of Biological Chemistry

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The sequence homology between Acanthamoeba myosin I heavy chain kinase (MIHCK) and other p21-activated kinases (PAKs) is relatively low, including only the catalytic domain and a short PAK N-terminal motif (PAN), and even these regions are not highly homologous. In this paper, we report the expression in insect cells of full-length, fully regulated Acanthamoeba MI-HCK and further characterize the regulation of this PAK by Rac, calmodulin, and autoinhibition. We map the autoinhibitory region of MIHCK to its PAN region and show that the PAN region inhibits autophosphorylation and kinase activity of unphosphorylated fulllength MIHCK and its expressed catalytic domain but has very little effect on either when they are phosphorylated. These properties are similar to those reported for mammalian PAK1. Unlike PAK1, MIHCK is activated by Rac only in the presence of phospholipid. However, peptides containing the PAN region of MIHCK bind Rac in the absence of lipid, and Rac binding reverses the inhibition of the MIHCK catalytic domain by PAN peptides. Our data suggest that a region N-terminal to PAN is required for optimal binding of Rac. Also unlike mammalian PAK, phospholipid stimulation of Acanthamoeba MIHCK and Dictyostelium MIHCK) (which is also a PAK) is inhibited by Ca 2؉ -calmodulin. In contrast to Dictyostelium MIHCK, however, Ca 2؉ -calmodulin also inhibits Rac-induced activity of Acanthamoeba MIHCK. The basic region N-terminal to PAN is essential for calmodulin binding. MIHCK1 phosphorylates a single Ser or Thr in the head domain of each of the three class-I myosins from Acanthamoeba castellanii substantially increasing their actin-activated MgATPase activities (for reviews, see Refs. 1 and 2). When the amino acid sequence of its catalytic domain is compared with the catalytic domains of other kinases (3), MIHCK is most similar to PAK1, a member of the PAK-I family of the mammalian Ste20 group kinases (according to the classification system of Dan et al. (4)).The PAK-I family (for reviews, see Refs. 5-8) share an homologous C-terminal catalytic domain and a conserved autoregulatory region (9) in the N-terminal half, also called PAN for PAK N-terminal motif (5, 10). This region is responsible for autoinhibition of mammalian PAK1, -2, and -3 (members of the PAK-I family) and for binding of Rac and Cdc42, which reverses autoinhibition (9 -14). As described by Lei et al. (9), the ϳ80-residue autoregulatory region of mammalian PAK1 consists of an N-terminal p21-binding domain (PBD) of ϳ44 residues, an overlapping inhibitory switch (IS) domain of ϳ50 residues that includes the C-terminal ϳ27 residues of the PBD and a C-terminal ϳ14-residue kinase inhibitory domain. A CRIB (Cdc42/Rac interactive binding) motif (15) of ϳ16 residues near the N terminus of the autoregulatory region is an essential component of the PBD. PAK4 (16) and other PAK-II family members also have a C-terminal catalytic domain and an N-terminal PBD but lack a recognizable autoinhibitory domain (4).Acanthamoeba MIHCK has a C-terminal catalytic domai...

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Structure of the Complex of Cdc42Hs with a Peptide Derived from P-21 Activated Kinase^,

Cited by 40 publications

References 61 publications

Conformational Switch and Role of Phosphorylation in PAK Activation

Conformational Switch and Role of Phosphorylation in PAK Activation

Effector Proteins Exert an Important Influence on the Signaling-active State of the Small GTPase Cdc42

Calmodulin-binding and Autoinhibitory Domains ofAcanthamoeba Myosin I Heavy Chain Kinase, a p21-activated Kinase (PAK)

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Structure of the Complex of Cdc42Hs with a Peptide Derived from P-21 Activated Kinase,

Cited by 40 publications

References 61 publications

Conformational Switch and Role of Phosphorylation in PAK Activation

Conformational Switch and Role of Phosphorylation in PAK Activation

Effector Proteins Exert an Important Influence on the Signaling-active State of the Small GTPase Cdc42

Calmodulin-binding and Autoinhibitory Domains ofAcanthamoeba Myosin I Heavy Chain Kinase, a p21-activated Kinase (PAK)

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About

Structure of the Complex of Cdc42Hs with a Peptide Derived from P-21 Activated Kinase^,