KSR modulates signal propagation within the MAPK cascade.

Abstract

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...

Isolation of a new protein factor required for activation of Raf-1 by Ha-Ras: partial purification from rat brain cytosols

“…Ksr-1 was originally identi®ed as a suppressor of abnormal phenotype induced by an activated mutant of Ras in both Drosophila (Therrien et al, 1995) and Caenorhabditis elegans (Kornfeld et al, 1995;Sundaram and Han, 1995). Since Ksr-1 associates with Raf-1 in a Rasdependent manner (Therrien et al, 1996), it may also be a candidate for a cytosolic Raf-1 activator.…”

Meaningful relationships: the regulation of the Ras/Raf/MEK/ERK pathway by protein interactions

“…This required the cysteine-rich CA3 domain in the N-terminal region, but not KSR kinase activity. In fact, the KSR kinase domain alone inhibited all these effects [111]. Further, KSR associated with Raf-1 at the cell membrane in a Ras-dependent manner and could enhance Raf-1 activation, a trait that was again traced to CA3, a cysteine-rich domain resembling the Raf CRD [112].…”

“…MEK binds to the kinase domain of KSR [121], which functions as a strong dominant-negative mutant [111,121] when severed from the Nterminus, presumably by sequestering MEK. The biochemical analysis of KSR protein interactions turned up a number of familiar faces, including Hsp90, Hsp70, Cdc37, MEK-1 and -2, and 14-3-3 [78].…”

Interactions of c-Raf-1 with phosphatidylserine and 14-3-3

“…At the membrane Raf-1 is activated partially by interactions with Ras (Drugan et al, 1996;Mineo et al, 1997;Roy et al, 1997;Stokoe and McCormick, 1997) but also by other mechanisms that require phosphorylation of tyrosine (Fabian et al, 1993;Dent et al, 1995a;Marais et al, 1995;Jelinek et al, 1996) and the presence of certain serine and threonine residues which may be phosphorylated (Fabian et al, 1993;Dent et al, 1995a;Diaz et al, 1997). Candidate kinases that may regulate Raf-1 activity at the plasma membrane include the sca old protein KSR (Yao et al, 1995;Therrien et al, 1996;Zhang et al, 1997), protein kinase C (Kolch et al, 1993), cAMP-dependent protein kinase or protein kinase A (Kikuchi and Williams, 1996) and AMP-activated protein kinase (Sprenkle et al, 1997). Activated Raf-1 phosphorylates and activates MEK1 and MEK2 which in turn phosphorylate and activate ERK1 and ERK2 (Marais and Marshall, 1996;Pritchard and McMahon, 1997).…”