Related Experiment Video

Updated: Aug 1, 2026

Oligopeptide Competition Assay for Phosphorylation Site Determination
09:16

Oligopeptide Competition Assay for Phosphorylation Site Determination

Published on: May 18, 2017

Phosphorylation of the Ras nucleotide exchange factor son of sevenless by mitogen-activated protein kinase

A D Cherniack1, J K Klarlund, M P Czech

  • 1Program in Molecular Medicine, University of Massachusetts Medical Center, Worcester 01605.

The Journal of Biological Chemistry
|February 18, 1994
PubMed

Insights

Growth factor signaling involves Son of sevenless (Sos) proteins, which are phosphorylated by mitogen-activated protein (MAP) kinases. This suggests a feedback loop where MAP kinases regulate Sos activity in cellular signaling pathways.

Area of Science:

  • Cellular signaling pathways
  • Molecular biology
  • Protein kinases

Background:

  • Son of sevenless (Sos)-1 and -2 are guanine nucleotide exchange factors crucial for Ras activation.
  • Ras signaling is initiated by insulin and epidermal growth factor (EGF) and activates mitogen-activated protein (MAP) kinases.
  • Sos proteins possess potential MAP kinase phosphorylation sites in their carboxyl-terminal regions.

Purpose of the Study:

  • To investigate whether MAP kinases phosphorylate Sos proteins.
  • To determine if identified phosphorylation sites are functional substrates for MAP kinases.

Main Methods:

  • Constructed a hemagglutinin (HA)-tagged Drosophila Sos (dSosHA) fusion protein.
  • Expressed dSosHA in COS-1 cells and performed immunoprecipitation using anti-HA antibodies.
  • Assessed in vitro phosphorylation of dSosHA by purified MAP kinase using [gamma-32P]ATP and analyzed by SDS-PAGE and 2D phosphopeptide mapping.

Main Results:

  • A 180 kDa phosphorylated band of dSosHA was observed upon incubation with MAP kinase and [gamma-32P]ATP.
  • This phosphorylation was specific to MAP kinase activity, as no band appeared without the kinase or in control immunoprecipitations.
  • Two-dimensional phosphopeptide analysis confirmed that in vitro phosphorylated sites matched those found in vivo, indicating functional phosphorylation.

Conclusions:

  • Mitogen-activated protein (MAP) kinases directly phosphorylate Son of sevenless (Sos) proteins.
  • These findings support a feedback mechanism where activated MAP kinases regulate Sos protein function.
  • This regulation is critical for controlling Ras-mediated signal transduction pathways activated by growth factors.

More Related Videos

Characterize Disease-related Mutants of RAF Family Kinases by Using a Set of Practical and Feasible Methods07:49

Characterize Disease-related Mutants of RAF Family Kinases by Using a Set of Practical and Feasible Methods

Published on: July 17, 2019

Isolation of Whole Cell Protein Lysates from Mouse Facial Processes and Cultured Palatal Mesenchyme Cells for Phosphoprotein Analysis07:26

Isolation of Whole Cell Protein Lysates from Mouse Facial Processes and Cultured Palatal Mesenchyme Cells for Phosphoprotein Analysis

Published on: April 1, 2022

Related Concept Videos

Phosphorylation01:02

Phosphorylation

The addition or removal of phosphate groups from proteins is the most common chemical modification that regulates cellular processes. These modifications can affect the structure, activity, stability, and localization of proteins within cells as well as their interactions with other proteins.
During phosphorylation, protein kinases transfer the terminal phosphate group of ATP to specific amino acid side chains of substrate proteins. Serine, threonine, and tyrosine are the most commonly...
The Ras Gene02:38

The Ras Gene

The Ras-gene-encoded proteins are regulators of signaling pathways controlling cell proliferation, differentiation, or cell survival. The Ras-gene family in humans constitutes three primary members—the HRas, NRas, and KRas. These genes code for four functionally distinct yet closely related proteins—the HRas, NRas, KRas4A, and KRas4B. The involvement of mutant Ras genes in human cancer was first discovered in 1982 and is among the most common causes of human tumorigenesis.
Ras is a superfamily...
Amplifying Signals via Enzymatic Cascade01:22

Amplifying Signals via Enzymatic Cascade

When a ligand binds to a cell-surface receptor, the receptor's intracellular domain changes shape, which may either activate its enzyme function or allow its binding to other molecules. The initial signal is amplified by most signal transduction pathways. This means that a single ligand molecule can activate multiple molecules of a downstream target. Proteins that relay a signal are most commonly phosphorylated at one or more sites, activating or inactivating the protein. Kinases catalyze the...
Small GTPases - Ras and Rho01:24

Small GTPases - Ras and Rho

Ras and Rho are small monomeric GTPases that act downstream of receptor tyrosine kinase (RTK) and regulate various cellular processes. These GTPases switch between active and inactive states by binding to guanine nucleotides.
Three regulatory proteins control their activity:
MAPK Signaling Cascades01:07

MAPK Signaling Cascades

Mitogen-activated protein kinase, or MAPK pathway, activates three sequential kinases to regulate cellular responses such as proliferation, differentiation, survival, and apoptosis. The canonical MAPK pathway starts with a mitogen or growth factor binding to an RTK. The activated RTKs stimulate Ras, which recruits Raf or MAP3 Kinase (MAPKKK), the first kinase of the MAPK signaling cascade. Raf further phosphorylates and activates MEK or MAP2 Kinases (MAPKK), which in turn phosphorylates MAP...
PI3K/mTOR/AKT Signaling Pathway01:22

PI3K/mTOR/AKT Signaling Pathway

The mammalian target of rapamycin  (mTOR) is a serine/threonine kinase that regulates growth, proliferation, and cell survival in response to hormones, growth factors, or nutrient availability. This kinase exists in two structurally and functionally distinct forms: mTOR complex 1  (mTORC1) and mTOR complex 2  (mTORC2). The first form (mTORC1) is composed of a rapamycin-sensitive Raptor and proline-rich Akt substrate, PRAS40. In contrast,  mTORC2 consists of a rapamycin-insensitive companion...