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...