Identification of the Mitogen-Activated Protein Kinase Phosphorylation Sites on Human Sosl That Regulate Interaction with Grb2

Abstract

The Son of sevenless proteins (Sos) are guanine nucleotide exchange factors involved in the activation of Ras by cytoplasmic and receptor tyrosine kinases. Growth factor stimulation rapidly induces the phosphorylation of Sos on multiple serine and threonine sites. Previous studies have demonstrated that growth factor-induced Sos phosphorylation occurs at the C-terminal region of the protein and is mediated, in part, by mitogen-activated protein (MAP) kinase. In this report, we describe the identification of five MAP kinase sites (S-1137, S-1167, S-1178, S-1193, and S-1197) on hSos1. We demonstrate that four of these sites, S-1132, S-1167, S-1178, and S-1193, become phosphorylated following growth factor stimulation. The MAP kinase phosphorylation sites are clustered within a region encompassing three proline-rich SH3-binding sites in the C-terminal domain of hSos1. Replacing the MAP kinase phosphorylation sites with alanine residues results in an increase in the binding affinity of Grb2 to hSos1. Interestingly, hSos2 contains only one MAP kinase phosphorylation site and, as demonstrated previously, has an increased affinity toward Grb2 compared with hSos1. These results suggest a role for MAP kinase in the regulation of Grb2-Sos interactions. Since the binding of Grb2 is important for Sos function, the phosphorylation-dependent modulation of Grb2-Sos association may provide a means of controlling Ras activation.

Cell cycle regulated expression and phosphorylation of hpttg proto-oncogene product

“…It is tempting to speculate that phosphorylation and dephosphorylation of Ser 165 would regulate the interaction of hPTTG with SH3-containing proteins. In fact, although SH3/prolines interactions were initially thought to be constitutive, there are examples of regulation of these interactions by phosphorylation (Buday et al, 1995;Corbalan-Garcia et al, 1996).…”

EGF induced SOS phosphorylation in PC12 cells involves P90 RSK-2

“…After overnight renaturation and kinase assay, two di erent growth factor inducible kinase activities were found on the SOS gel; a kinase doublet at 43 kDa, and a kinase at approximately 90 kDa (Figure 1a). The kinases at 43 kDa, present in both the SOS gel and the MBP gel ( Figure 1a and b), are likely to be p42 Erk-2 and p44 Erk-1 as these growth factor inducible kinases are known to utilize both these proteins as substrates (Cherniack et al, 1994;Corbalan-Garcia et al, 1996b;Rozakis-Adcock et al, 1995;Boulton et al, 1991). In contrast, the 90 kDa growth factor inducible kinase was only seen in the SOS gel and therefore appeared to be speci®c for this substrate.…”

“…Results obtained using speci®c inhibitors of the MAP kinase kinase, MEK (Langlois et al, 1995;Waters et al, 1995a), or cAMP which blocks MAPK activation in response to growth factors (Burgering et al, 1993;Corbalan-Garcia et al, 1996a), would indicate that the p42 and p44 MAP kinases are most likely responsible for SOS phosphorylation. Consistent with these observations, the proline-rich carboxy-terminal region of SOS contains a number of MAPK consensus sites, some of which have been shown to be phosphorylated by this kinase both in vitro and in vivo (Cherniack et al, 1994;Corbalan-Garcia et al, 1996b;Rozakis-Adcock et al, 1995). By analogy to S. cerivisiae where Cdc25 phosphorylation is involved in down regulation of the Ras pathway (Gross et al, 1992), MAP kinase phosphorylation of SOS in mammalian cells is thought to play a similar role.…”

“…However, while no change in SOS exchange activity has yet been demonstrated in response to MAP kinase phosphorylation, the binding to Grb2 and Shc has been shown to be altered. Indeed in response to di erent stimuli such as insulin or EGF, the resulting phosphorylation of SOS is thought to play a negative feedback role on the Ras pathway, by causing the dissociation of Grb2 from SOS (Corbalan-Garcia et al, 1996a;Waters et al, 1995b;Cherniack et al, 1995) or alternatively, dissociation of the Grb2-SOS binary complex from the receptor (Rozakis- Adcock et al, 1995;Klarlund et al, 1995;Langlois et al, 1995;Por®ri and McCormick, 1996). While MAP kinase phosphorylation may partially be responsible for these e ects on SOS, recently it has been shown that a kinase other than MAP kinase is also involved in SOS phosphorylation.…”

“…While MAP kinase phosphorylation may partially be responsible for these e ects on SOS, recently it has been shown that a kinase other than MAP kinase is also involved in SOS phosphorylation. Furthermore in vivo phosphopeptide mapping of SOS has indicated that not all sites can be accounted for by MAP kinase phosphorylation (Corbalan-Garcia et al, 1996b;Rozakis-Adcock et al, 1995). In addition, Holt et al have also shown by expressing MKP-1, the MAPK phosphatase, that a kinase other than MAPK can induce a mobility shift in SOS as well as its dissociation from Grb2 in response to insulin (Holt et al, 1996a).…”