Genetic studies have implicated the cytosolic juxtamembrane region of the Kit receptor tyrosine kinase as an autoinhibitory regulatory domain. Mutations in the juxtamembrane domain are associated with cancers, such as gastrointestinal stromal tumors and mastocytosis, and result in constitutive activation of Kit. Here we elucidate the biochemical mechanism of this regulation. A synthetic peptide encompassing the juxtamembrane region demonstrates cooperative thermal denaturation, suggesting that it folds as an autonomous domain. The juxtamembrane peptide directly interacted with the N-terminal ATP-binding lobe of the kinase domain. A mutation in the juxtamembrane region corresponding to an oncogenic form of Kit or a tyrosine-phosphorylated form of the juxtamembrane peptide disrupted the stability of this domain and its interaction with the N-terminal kinase lobe. Kinetic analysis of the Kit kinase harboring oncogenic mutations in the juxtamembrane region displayed faster activation times than the wild-type kinase. Addition of exogenous wild-type juxtamembrane peptide to active forms of Kit inhibited its kinase activity in trans, whereas the mutant peptide and a phosphorylated form of the wild-type peptide were less effective inhibitors. Lastly, expression of the Kit juxtamembrane peptide in cells which harbor an oncogenic form of Kit inhibited cell growth in a Kit-specific manner. Together, these results show the Kit kinase is autoinhibited through an intramolecular interaction with the juxtamembrane domain, and tyrosine phosphorylation and oncogenic mutations relieved the regulatory function of the juxtamembrane domain.Receptor tyrosine kinases (RTKs) activate intracellular signaling pathways that control cellular growth, differentiation, and metabolism. The catalytic activity of RTKs is tightly controlled through a number of mechanisms, including ligand binding, internalization and degradation, and the activation of protein tyrosine phosphatases (14, 37). Disruption of any of these control points can lead to constitutive receptor activation and subsequent cellular transformation.Recently, mutations in Kit which result in ligand-independent activation of the kinase were found to be associated with human gastrointestinal stromal tumors (GISTs) (15) and mastocytosis (11). Kit mutations in GISTs most frequently occur in the noncatalytic juxtamembrane (JM) region, suggesting that this region is crucial in regulation of kinase activity (28). GIST JM mutations are comprised of deletions, substitutions, a combination of deletions and substitutions, or tandem duplications. The retroviral version of Kit originally identified in a feline sarcoma retroviral complex also has mutations and deletions in the JM region (3).Two other members of the type III RTK family, plateletderived growth factor receptor ␤ (PDGFR␤) and Flt3, have been reported to contain activating mutations in their JM regions (13,19,29,32). Similar to Kit, these mutations result in ligand-independent kinase activation. The Flt3 JM mutations are tandem duplic...

Background: CRMPs play roles in axon specification and semaphorin 3A-induced growth cone collapse, but their biochemical function is unclear. Results: CRMPs are found to bind directly to microtubules through a conserved C-terminal region. Conclusion: CRMPs can stabilize microtubules but are negatively regulated by phosphorylation. Significance: This work can explain phenotypes associated with loss of CRMPs on axon specification and dendritic arborization.

Fractionation of brain extracts and functional biochemical assays identified PP2C␣, a serine/threonine phosphatase, as the major biochemical activity inhibiting PAK1. PP2C␣ dephosphorylated PAK1 and p38, both of which were activated upon hyperosmotic shock with the same kinetics. In comparison to growth factors, hyperosmolality was a more potent activator of PAK1. Therefore we characterize the PAK signaling pathway in the hyperosmotic shock response. Endogenous PAKs were recruited to the p38 kinase complex in a phosphorylation-dependent manner. Overexpression of a PAK inhibitory peptide or dominant negative Cdc42 revealed that p38 activation was dependent on PAK and Cdc42 activities. PAK mutants deficient in binding to Cdc42 or PAK-interacting exchange factor were not activated. Using a panel of kinase inhibitors, we identified PI3K acting upstream of PAK, which correlated with PAK repression by pTEN overexpression. RNA interference knockdown of PAK expression reduced stress-induced p38 activation and conversely, PP2C␣ knockdown increased its activation. Hyperosmotic stressinduced PAK translocation away from focal adhesions to the perinuclear compartment and resulted in disassembly of focal adhesions, which are hallmarks of PAK activation. Inhibition of PAK by overexpression of PP2C␣ or the kinase inhibitory domain prevented sorbitol-induced focal adhesion dissolution. Inhibition of MAPK pathways showed that MEK-ERK signaling but not p38 is required for full PAK activation and focal adhesion turnover. We conclude that 1) PAK plays a required role in hyperosmotic signaling through the PI3K/pTEN/Cdc42/PP2C␣/p38 pathway, and 2) PAK and PP2C␣ modulate the effects of this pathway on focal adhesion dynamics. PAK,2 the p21-activated kinase, is an effector kinase for the small Rho GTPases Cdc42 and Rac (1). PAKs mediate cytoskeletal rearrangements promoted by the activated GTPases such as loss of focal adhesions and actin stress fibers and the generation of filopodia (2, 3). PAK has also been implicated in other cellular events, including protection from apoptosis through phosphorylation of BAD (4, 5), mitosis through phosphorylation of RAF-1 (6, 7), and hormone signaling through estrogen receptor phosphorylation (8). The mitogen-activated protein kinase (MAPK) pathway is linked to PAK through Cdc42-mediated activation of p38, JNK (9), and ERK (10). The signaling pathways of extracellular stimuli leading to PAK and MAPK activation are not well characterized.Changes in extracellular osmolality rapidly induce the activation of MAPKs (11); however, little is known of the regulators of the MAPK pathway. In Saccharomyces cerevisiae, stress response is mediated through specific osmosensing pathways, of which components include the MAPKs (12). The mammalian counterpart of these osmosensors has not been conclusively identified, although clustering of epidermal growth factor receptor has been proposed (13). PAK has been implicated in the stress response pathway through its activation upon hyperosmotic shock (14, 15).The mechanis...

ACK1 (activated Cdc42-associated kinase 1), a cytoplsmic tyrosine kinase, is implicated in metastatic behavior, cell spreading and migration, and epidermal growth factor receptor (EGFR) signaling. The function of ACK1 in the regulation of receptor tyrosine kinases requires a C-terminal region that demonstrates a significant homology to the EGFR binding domain of MIG6. In this study, we have identified additional receptor tyrosine kinases, including Axl, leukocyte tyrosine kinase, and anaplastic lymphoma kinase, that can bind to the ACK1/MIG6 homology region. Unlike the interaction between MIG6 and EGFR, our data suggest that these receptor tyrosine kinases require the adaptor protein Grb2 for efficient binding, which interacts with highly conserved proline-rich regions that are conserved between ACK1 and MIG6. We have focused on Axl and compared how ACK1/Axl differs from the ACK1/EGFR axis by investigating effects of knockdown of endogenous ACK1. Although EGFR activation promotes ACK1 turnover, Axl activation by GAS6 does not; interestingly, the reciprocal down-regulation of GAS6-stimulated Axl is blocked by removing ACK1. Thus, ACK1 functions in part to control Axl receptor levels. Silencing of ACK1 also leads to diminished ruffling and migration in DU145 and COS7 cells upon GAS6-Axl signaling. The ability of ACK1 to modulate Axl and perhaps anaplastic lymphoma kinase (altered in anaplastic large cell lymphomas) might explain why ACK1 can promote metastatic and transformed behavior in a number of cancers.

Crystal structures of inactive PAK1(K299R) and the activation (A)-loop phospho-mimetic PAK1(T423E) have suggested that the kinase domain is in an active state regardless of activation loop status. Contrary to a large body of literature, we find that neither is PAK1(T423E) active in cells, nor does it exhibit significant activity in vitro. To explain these discrepancies all-atom molecular dynamics (MD) simulations of PAK1(phospho-T423) in complex with ATP and substrate were performed. These simulations point to a key interaction between PAK1 Lys308, at the end of the alphaC helix, and the pThr423 phosphate group, not seen in X-ray structures. The orthologous PAK4 Arg359 fulfills the same role in immobilizing the alphaC helix. These in silico predictions were validated by experimental mutagenesis of PAK1 and PAK4. The simulations explain why the PAK1 A-loop phospho-mimetic is inactive, but also point to a key functional interaction likely found in other protein kinases.

c-Abl is a non-receptor tyrosine kinase that is involved in a variety of signaling pathways. Activated forms of c-Abl are associated with some forms of human leukemia. Presently, no high resolution structure of the tyrosine kinase domain of Abl is available. We have developed a structural homology model of the catalytic domain of Abl based on the crystal structure of the insulin receptor tyrosine kinase. Using this model as a guide, we selected residues near the active site predicted to play a role in peptide/protein substrate recognition. We expressed and purified 15 mutant forms of Abl with single amino acid substitutions at these positions and tested their peptide substrate specificity. We report here the identification of seven residues involved in recognition of the P؊1, P؉1, and P؉3 positions of bound peptide substrate. Mutations in these residues cause distinct changes in substrate specificity. The results suggest features of Abl substrate recognition that may be relevant to related tyrosine kinases.The c-abl proto-oncogene encodes a multidomain non-receptor tyrosine kinase that is expressed ubiquitously in human tissues (reviewed in Refs. 1-4). Mutant forms of c-abl are found in patients with Philadelphia chromosome-positive chronic myelogenous leukemia and acute lymphocytic leukemia (1-4). In these diseases, a chromosomal translocation event produces a chimeric oncogene consisting of 5Ј-sequences of bcr fused to abl. The BCR-Abl fusion protein has elevated tyrosine kinase activity relative to c-Abl, and the tyrosine kinase activity of the BCR-Abl fusion protein is necessary for disease progression. Similarly, tyrosine kinase activity is necessary for transformation of fibroblasts or hematopoietic cells by BCR-Abl (5, 6).In addition to its tyrosine kinase catalytic domain, c-Abl has a short amino-terminal unique domain followed by SH3 and SH2 domains (1-4). This domain organization is found in many non-receptor tyrosine kinases. Abl also possesses a large carboxyl-terminal region that includes a DNA-binding domain, an F-actin-binding domain, a nuclear localization signal, and a proline-rich region implicated in mediating protein-protein interactions. Studies aimed at understanding the normal physiological role of c-Abl have shown the enzyme to be involved in signal transduction, cytoskeletal rearrangement, RNA polymerase II activation, DNA repair, and cell cycle control (1-4). c-Abl has been shown to physically associate with at least seven unique proteins, including p53 and the nuclear Rb protein (4). Mice with targeted disruptions in the c-abl gene have high neonatal mortality rates and are more susceptible to infection, suggesting a role for c-abl in B-lymphocyte development (7).At least eight in vivo substrates for Abl have been identified (4). The amino acid sequences surrounding the phosphorylation sites for two of these proteins, RNA polymerase II (8) and c-Crk (9), have been described. These sequences do not share a common primary sequence motif, suggesting that Abl may have a broad range of subs...

Receptor tyrosine kinases couple a wide variety of extracellular cues to cellular responses. The class III subfamily comprises the platelet-derived growth factor receptor, c-Kit, Flt3 and c-Fms, all of which relay cell proliferation signals upon ligand binding. Accordingly, mutations in these proteins that confer ligand-independent activation are found in a subset of cancers. These mutations cluster in the juxtamembrane (JM) and catalytic tyrosine kinase domain (TKD) regions. In the case of acute myeloid leukemia (AML), the juxtamembrane (named ITD for internal tandem duplication) and TKD Flt3 mutants differ in their spectra of clinical outcomes. Although the mechanism of aberrant activation has been largely elucidated by biochemical and structural analyses of mutant kinases, the differences in disease presentation cannot be attributed to a change in substrate specificity or signaling strength of the catalytic domain. This review discusses the latest literature and presents a working model of differential Flt3 signaling based on mis-localized juxtamembrane autophosphorylation, to account for the disease variation. This will have bearing on therapeutic approaches in a complex disease such as AML, for which no efficacious drug yet exists.

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