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Although RAF kinases are critical for controlling cell growth, their mechanism of activation is incompletely understood. Recently, dimerization was shown to be important for activation. Here we show that the dimer is functionally asymmetric with one kinase functioning as an activator to stimulate activity of the partner, receiver kinase. The activator kinase did not require kinase activity but did require N-terminal phosphorylation that functioned allosterically to induce cis-autophosphorylation of the receiver kinase. Based on modeling of the hydrophobic spine assembly, we also engineered a constitutively active mutant that was independent of Ras, dimerization, and activation-loop phosphorylation. As N-terminal phosphorylation of BRAF is constitutive, BRAF initially functions to activate CRAF. N-terminal phosphorylation of CRAF was dependent on MEK, suggesting a feedback mechanism and explaining a key difference between BRAF and CRAF. Our work illuminates distinct steps in RAF activation that function to assemble the active conformation of the RAF kinase.

A study showed that CD2 binding to ligand results to protein segregation, CD2 clustering and T cell polarization. The protein segregation is independent of the cytoplasmic domain of CD2, while the CD2 clustering and cytoskeleton polarization are mediated by binding of the CD2 cytoplasmic domain with a new SH3-containing protein called CD2-associated protein (CD2AP). These indicate that the CD2AP seems to promote receptor patterning and cytoskeletal polarization in the T cell contact area.

Analysis of intracellular signal transduction after activation by anergy-inducing altered peptide ligands (APLs) and of the phosphorylation patterns reveals that anergy-inducing APL does not cause protein-tyrosine phosphorylation, indicating the stimulation of selective signal transduction pathways by APL. APL stimulation induces a characteristic T cell receptor (TCR) zeta chain phosphorylation and inhibits its correlation to the zap70 protein-tyrosine kinase. The TCR-CD3 complex thus associates with selective intracellular biochemical signaling pathways, resulting in anergic T cell phenotype.

The role of plasma-membrane microdomains in the organization of signaling proteins is a controversial issue in T cell signaling. Single-molecule fluorescence is used to suggest that protein-protein interactions, not detergent insolubility, regulate the assembly of signaling complexes in the plasma membrane.