Nck is an adaptor protein composed of a single SH2 domain and three SH3 domains. Upon growth factor stimulation, Nck is recruited to receptor tyrosine kinases via its SH2 domain, probably initiating one or more signaling cascades. In this report, we show that Nck is bound in living cells to the serine-threonine kinase Pak1. The association between Nck and Pak1 is mediated by the second SH3 domain of Nck and a proline-rich sequence in the amino terminus of Pak1. We also show that Pak1 is recruited by activated epidermal growth factor (EGF) and platelet-derived growth factor receptors. Moreover, Pak1 kinase activity is increased in response to EGF in HeLa cells transfected with human Pak1, and the kinase activity was enhanced when Nck was co-transfected. It is concluded that Nck links receptor tyrosine kinases with Pak1 and is probably involved in targeting and regulation of Pak1 activity.

The murine retroviral oncogene v-cbl induces pre-B cell lymphomas and myelogenous leukemias. The protein product of the mammalian c-cbl proto-oncogene is a widely expressed cytoplasmic 120-kDa protein (p120cbl) whose normal cellular function has not been determined. Here we show that upon stimulation of human epidermal growth factor (EGF) receptor, p12ocbl becomes strongly tyrosine-phosphorylated and associates with activated EGF receptor in vivo. A GST fusion protein containing amino acids 1-486 of p120cbl, including a region highly conserved in nematodes, binds directly to the autophosphorylated carboxyl-terminal tail of the EGF receptor. Platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), or nerve growth factor (NGF) stimulation also results in tyrosine phosphorylation of p120cbl. Recent genetic studies in Caenorhabditis elegans indicate that Sli-1, a p120cbl homologue, plays a negative regulatory role in control of the Ras signaling pathway initiated by the C. elegans EGF receptor homologue. Our results indicate that p120cbl is involved in an early step in the EGF signaling pathway that is conserved from nematodes to mammals.

Ack͞Ack1 is a nonreceptor protein tyrosine kinase that comprises a tyrosine kinase core, an SH3 domain, a Cdc42-binding region, a Ralt homology region, and a proline-rich region. Here we describe a detailed characterization of the Ack protein as well as the chromosomal localization of human Ack (chromosome 3q29) and the primary structure of murine Ack. We demonstrate that Ack is ubiquitously expressed, with highest expression seen in thymus, spleen, and brain. Activation of integrins by cell adhesion on fibronectin leads to strong tyrosine phosphorylation and activation of Ack. Upon cell stimulation with EGF or PDGF, Ack is tyrosine-phosphorylated and recruited to activated EGF or PDGF receptors, respectively. A pool of endogenous Ack molecules is constitutively tyrosine-phosphorylated, even in starved cells. Moreover, tyrosine-phosphorylated Ack forms a stable complex with the adapter protein Nck via its SH2 domain. Finally, we have characterized a membrane-targeting sterile ␣ motif-like domain in the amino terminus of Ack. Using several Ack mutants, we show that the amino-terminal and CRIB domains are necessary for Ack autophosphorylation, whereas the SH3 domain appears to have an autoinhibitory role. These experiments suggest a functional role for Ack as an early transducer of multiple extracellular stimuli.cell signaling ͉ tyrosine phosphorylation ͉ surface receptors ͉ growth factors ͉ cell adhesion

The epidermal growth factor receptor (EGFR) and the non-receptor protein tyrosine kinases Src and Pyk2 have been implicated in linking a variety of G-proteincoupled receptors (GPCR) to the mitogen-activated protein (MAP) kinase signaling cascade. In this report we apply a genetic strategy using cells isolated from Src-, Pyk2-, or EGFR-deficient mice to explore the roles played by these protein tyrosine kinases in GPCR-induced activation of EGFR, Pyk2, and MAP kinase. We show that Src kinases are critical for activation of Pyk2 in response to GPCR-stimulation and that Pyk2 and Src are essential for GPCR-induced tyrosine phosphorylation of EGFR. By contrast, Pyk2, Src, and EGFR are dispensable for GPCR-induced activation of MAP kinase. Moreover, GPCR-induced MAP kinase activation is normal in fibroblasts deficient in both Src and Pyk2 (Src؊/؊Pyk2؊/؊ cells) as well as in fibroblasts deficient in all three Src kinases expressed in these cells (Src؊/ ؊Yes؊/؊Fyn؊/؊ cells). Finally, experiments are presented demonstrating that, upon stimulation of GPCR, activated Pyk2 forms a complex with Src, which in turn phosphorylates EGFR directly. These experiments reveal a role for Src kinases in Pyk2 activation and a role for Pyk2 and Src in tyrosine phosphorylation of EGFR following GPCR stimulation. In addition, EGFR, Src family kinases, and Pyk2 are not required for linking GPCRs with the MAP kinase signaling cascade.

During the packaging of double-stranded DNA by bacterial viruses, the precursor procapsid loses its internal core of scaffolding protein and undergoes a substantial expansion to form the mature virion. Here we show that upon heating, purified P22 procapsids release their scaffolding protein subunits, and the coat protein lattice expands in the absence of any other cellular or viral components. Following these processes by differential scanning calorimetry revealed four different transitions that correlated with structural transitions in the coat protein shells. Exit of scaffolding protein from the procapsid occurred reversibly and just above physiological temperature. Expansion of the procapsid lattice, which was exothermic, occurred after the release of scaffolding protein. Partial denaturation of coat subunits within the intact shell structure was detected prior to the major endothermic event. This major endotherm occurred above 80 degrees C and represents particle breakage and irreversible coat protein denaturation. The results indicate that the coat subunits are designed to form a metastable precursor lattice, which appears to be separated from the mature lattice by a kinetic barrier.

In the presence of guanidine hydrochloride, phosphoglycerate kinase from yeast can be reversibly denatured by either heating or cooling the protein solution above or below room temperature [Griko, Y. V., Venyaminov, S. Y., & Privalov, P. L. (1989) FEBS Lett. 244, 276-278]. The heat denaturation of PGK is characterized by the presence of a single peak in the excess heat capacity function obtained by differential scanning calorimetry. The transition curve approaches the two-state mechanism, indicating that the two domains of the molecule display strong cooperative interactions and that partially folded intermediates are not largely populated during the transition. On the contrary, the cold denaturation is characterized by the presence of two peaks in the heat capacity function. Analysis of the data indicates that at low temperatures the two domains behave independently of each other. The crystallographic structure of PGK has been used to identify the nature of the interactions between the two domains. These interactions involve primarily the apposition of two hydrophobic surfaces of approximately 480 A2 and nine hydrogen bonds. This information, in conjunction with experimental thermodynamic values for hydrophobic, hydrogen bonding interactions and statistical thermodynamic analysis, has been used to quantitatively account for the folding/unfolding behavior of PGK. It is shown that this type of analysis accurately predicts the cooperative behavior of the folding/unfolding transition and its dependence on GuHCl concentration.

Differential scanning calorimetry transitions for the irreversible thermal denaturation of yeast phosphoglycerate kinase at pH 7.0 are strongly scanning-rate dependent, suggesting that the denaturation is, at least in part, under kinetic control. To test this possibility, we have carried out a kinetic study on the thermal inactivation of the enzyme. The inactivation kinetics are comparatively fast within the temperature range of the calorimetric transitions and can be described phenomenologically by the equation dC/dt = -alpha C2/(beta + C), where C is the concentration of active enzyme at a given time, t, and alpha and beta are rate coefficients that depend on temperature. This equation, together with the values of alpha and beta (within the temperature range 50-59 degrees C) have allowed us to calculate the fraction of irreversibly denatured protein versus temperature profiles corresponding to the calorimetric experiments. We have found that (a) irreversible denaturation takes place during the time the protein spends in the transition region and (b) there is an excellent correlation between the temperatures of the maximum of the calorimetric transitions (Tm) and the temperatures (Th) at which half of the protein is irreversibly denatured. These results show that the differential scanning calorimetry transitions for the denaturation of phosphoglycerate kinase are highly distorted by the rate-limited irreversible process. Finally, some comments are made as to the use of equilibrium thermodynamics in the analysis of irreversible protein denaturation.

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