Kinase suppressor of Ras (KSR) is a recently identified component of Ras-dependent signaling pathways. In this report, we show that murine KSR1 (mKSR1) cooperates with activated Ras to promote Xenopus oocyte maturation and cellular transformation and provide evidence that this cooperation occurs by accelerating mitogen and extracellular regulated kinase (MEK) and mitogen-activated protein kinase (MAPK) activation. We also find that mKSR1 associates with Raf-1 at the plasma membrane in a Ras-dependent manner, indicating the presence of a membrane-bound kinase signaling complex. Although mKSR1 is related structurally to Raf-1, our findings reveal striking functional differences between these proteins. In marked contrast to the isolated amino-and carboxy-terminal domains of Raf-1, the KSR amino terminus also cooperates with Ras, whereas the carboxy-terminal kinase domain blocks Ras signaling as well as MEK and MAPK activation. The isolated KSR kinase domain suppressed Xenopus oocyte maturation, cellular transformation, and Drosophila eye development, suggesting that separation of the amino-and carboxy-terminal domains has uncoupled the normal regulation of KSR as a positive effector of Ras signaling. Together, our findings indicate that mKSR1 is an integral component of the MAPK module functioning via a novel mechanism to modulate signal propagation between Raf~ MEK1, and MAPK.[Key Words: KSR; MAPK; MEK; Raf; Ras; signal transduction] Received August 1, 1996; revised version accepted September 9, 1996.Ras plays a central role in the transmission of proliferative and differentiative signals elicited by receptor tyrosine kinases (RTKs). A major route by which Ras-mediated signals are transduced from the plasma membrane to the nucleus involves the mitogen-activated protein kinase (MAPK) module (for review, see Marshall 1995). This module is composed of at least three kinases, Raf-1, MEK1 (MAPK kinase), and MAPK, which function to transmit cellular signals through a phosphorylation cascade. The current model proposes that upon Ras activation, Raf-1 is recruited to the plasma membrane by a direct association with Ras. Once at the membrane, Raf-1 becomes activated and promotes signal propagation by the sequential activation of MEK1 and MAPK (for review, see Moodie and Wolfman 1994). Activated MAPK then phosphorylates critical cytoplasmic and nuclear substrates, thereby regulating their activity and orchestrating a specific cellular response (for review, see Waskiewicz and Cooper 1995;Treisman 1996).Although this linear pathway provides a coherent model for how RTK-mediated signals are propagated from Ras to the nucleus, there are several important aspects of this process that remain to be resolved. In particular, the precise mechanism by which Raf-1 becomes activated at the membrane is unclear (Leevers et al. 1994;Stokoe et al. 1994). In addition, the mechanisms that provide signal specificity or that are required for signal termination within the MAPK module are largely unknown. Furthermore, other Ras-dependent pathways...

Recent reports have demonstrated the in vivo association of Raf-1 with members of the 14-3-3 protein family. To address the significance of the Raf-1-14-3-3 interaction, we investigated the enzymatic activity and biological function of Raf-1 in the presence and absence of associated 14-3-3. The interaction between these two molecules was disrupted in vivo and in vitro with a combination of molecular and biochemical techniques. Biochemical studies demonstrated that the enzymatic activities of Raf-1 were equivalent in the presence and absence of 14-3-3. Furthermore, mixing of purified Raf-1 and 14-3-3 in vitro was not sufficient to activate Raf-1. With a molecular approach, Cys-165 and Cys-168 as well as Ser-259 were identified as residues of Raf-1 required for the interaction with 14-3-3. Cys-165 and Cys-168 are located within the conserved cysteine-rich region of the CR1 domain, and Ser-259 is a conserved site of serine phosphorylation found within the CR2 domain. Mutation of either Cys-165 and Cys-168 or Ser-259 prevented the stable interaction of Raf-1 with 14-3-3 in vivo. Consistent with the model in which a site of serine phosphorylation is involved in the Raf-1-14-3-3 interaction, dephosphorylated Raf-1 was unable to associate with 14-3-3 in vitro. Phosphorylation may represent a general mechanism mediating 14-3-3 binding, because dephosphorylation of the Bcr kinase (known to interact with 14-3-3) also eliminated its association with 14-3-3. Finally, mutant Raf-1 proteins unable to stably interact with 14-3-3 exhibited enhanced enzymatic activity in human 293 cells and Xenopus oocytes and were biologically activated, as demonstrated by their ability to induce meiotic maturation of Xenopus oocytes. However, in contrast to wild-type Raf-1, activation of these mutants was independent of Ras. Our results therefore indicate that interaction with 14-3-3 is not essential for Raf-1 function.

Genetic and biochemical studies have identified kinase suppressor of Ras (KSR) to be a conserved component of Ras-dependent signaling pathways. To better understand the role of KSR in signal transduction, we have initiated studies investigating the effect of phosphorylation and protein interactions on KSR function. Here, we report the identification of five in vivo phosphorylation sites of KSR. In serum-starved cells, KSR contains two constitutive sites of phosphorylation (Ser297 and Ser392), which mediate the binding of KSR to the 14-3-3 family of proteins. In the presence of activated Ras, KSR contains three additional sites of phosphorylation (Thr260, Thr274, and Ser443), all of which match the consensus motif (Px[S/T]P) for phosphorylation by mitogen-activated protein kinase (MAPK). Further, we find that treatment of cells with the MEK inhibitor PD98059 blocks phosphorylation of the Ras-inducible sites and that activated MAPK associates with KSR in a Ras-dependent manner. Together, these findings indicate that KSR is an in vivo substrate of MAPK. Mutation of the identified phosphorylation sites did not alter the ability of KSR to facilitate Ras signaling in Xenopus oocytes, suggesting that phosphorylation at these sites may serve other functional roles, such as regulating catalytic activity. Interestingly, during the course of this study, we found that the biological effect of KSR varied dramatically with the level of KSR protein expressed. In Xenopus oocytes, KSR functioned as a positive regulator of Ras signaling when expressed at low levels, whereas at high levels of expression, KSR blocked Ras-dependent signal transduction. Likewise, overexpression of Drosophila KSR blocked R7 photoreceptor formation in the Drosophila eye. Therefore, the biological function of KSR as a positive effector of Ras-dependent signaling appears to be dependent on maintaining KSR protein expression at low or nearphysiological levels.Ras is a small, evolutionarily conserved GTPase that functions in the transmission of signals mediating cellular growth, development, and differentiation (4,26,27,29). Because Ras has been shown to play a critical role in both normal and abnormal growth processes, considerable research effort has focused on elucidating the components involved in and the mechanisms regulating Ras-dependent signal transduction. By a series of genetic and biochemical studies, a conserved pathway involving cell surface receptors, guanine nucleotide exchange factors, Ras, and serine/threonine kinases has been revealed (reviewed in references 23, 25, 29, and 42). In response to many growth and developmental stimuli, signals are initiated at the cell surface by the activation of receptor tyrosine kinases. Through the recruitment of guanine nucleotide exchange factors to the plasma membrane, receptor activation results in the conversion of Ras from the inactive GDP-bound form to the active GTP-bound form. Activated Ras then propagates the signal by interacting with its effector molecules, one of which is the Raf-1 serine/thr...

following correction should be noted. Due to an editorial change at PNAS, the meaning of the last sentence on page 14046 was altered. The sentence originally read as follows: On the other hand, this structure does not reproduce the pharmacological properties of either P or Q channel exactly, as the ID 50 to sFTX and -Aga IVA for P-type channels is lower than for the ␣1A, ␣2␦, ␤Ib channels in HEK cells.Neurobiology. In the article "The synthesis of ATP by glycolytic enzymes in the postsynaptic density and the effect of endogenously generated nitric oxide" Kuo Wu, Chiye Aoki, Alice Elste, Adrienne A. Rogalski-Wilk, and Philip Siekevitz, which appeared in number 24, November 25, 1997, of Proc. Natl. Acad. Sci. USA (94,(13273)(13274)(13275)(13276)(13277)(13278), the quality of the reproduction of Fig. 2A was poor. The figure and its legend are shown below:Biochemistry. In the article "KSR stimulates Raf-1 activity in a kinase-independent manner" by Neil R. Michaud, Marc Therrien, Angela Cacace, Lisa C. Edsall, Sarah Spiegel, Gerald M. Rubin, and Deborah K. Morrison, which appeared in number 24, November 25, 1997, of Proc. Natl. Acad. Sci. USA (94,(12792)(12793)(12794)(12795)(12796), the following correction should be noted.Due to a printer's error, background was incorrectly added to (50 g) and 100 g each of the other fractions, in 100 l final volume, including whole homogenate (H), synaptosomes (Syn), synaptic plasma membranes (SPM), and crude synaptic vesicles (CSV), were incubated at 37°C for 15 min. NAD incorporation was performed in the absence (Ϫ) or presence (ϩ) of SNP as exogenous source of NO. The mixtures were subjected to SDS͞PAGE and then autoradiography. (B) Western blot analysis of the G3PD in the subcellular fractions. To confirm that the radioactive protein in the subcellular fractions was indeed G3PD, Western blot analysis was performed by using specific anti-G3PD antibodies as described.