The kinome specific co-chaperone, CDC37 (cell division cycle 37), is responsible for delivering BRAF (B-Rapidly Accelerated Fibrosarcoma) to the Hsp90 (heat shock protein 90) complex, where it is then translocated to the RAS (protooncogene product p21) complex at the plasma membrane for RAS mediated dimerization and subsequent activation. We identify a bipartite interaction between CDC37 and BRAF and delimitate the essential structural elements of CDC37 involved in BRAF recognition. We find an extended and conserved CDC37 motif, 20HPNID---SL--W31, responsible for recognizing the C-lobe of BRAF kinase domain, while the c-terminal domain of CDC37 is responsible for the second of the bipartite interaction with BRAF. We show that dimerization of BRAF, independent of nucleotide binding, can act as a potent signal that prevents CDC37 recognition and discuss the implications of mutations in BRAF and the consequences on signaling in a clinical setting, particularly for class 2 BRAF mutations.
The kinome specific co-chaperone, CDC37, is responsible for delivering BRAF to the Hsp90 complex, where it is then translocated to the RAS complex at the plasma membrane for RAS mediated dimerization and subsequent activation. We identify a bipartite interaction between CDC37 and BRAF and delimitate the essential structural elements of CDC37 involved in BRAF recognition. We find an extended and conserved CDC37 motif, 20HPNID---SL--W31, responsible for recognising the C-lobe of BRAF kinase domain, while the C-terminal domain of CDC37 is responsible for the second of the bipartite interaction with BRAF. We show that dimerization of BRAF, independent of nucleotide binding, can act as a potent signal that prevents CDC37 recognition and discuss the implications of mutations in BRAF and the consequences on signalling, particularly for class 2 BRAF mutations.
Methylation and demethylation of Lysine and Arginine on proteins is quantitatively an extensive and functionally a significant post-translational modification yet there is a dearth of information at a functional systems level. Using SILAC proteomics and high resolution mass spectrometry we have identified eight demethylase and two methylase proteins whose levels are regulated during myogenic differentiation in a mouse myoblast-to-myocyte model. Using the general methylation inhibitor adenosine dialdehyde (AdOX) we established that methylation is required for differentiation. Whole proteome analysis revealed that 1134 out of 4600 proteins identified were differentially expressed, of which 488 were up-regulated and 646 down-regulated. Of these, two were methylases and eight were demethylases. Notably, five of the eight enzymes demethylate Lysine 9 on histone 3 (H3K9) whereas two also demethylate H3K4. Lastly, we have identified short linear motifs (SliMs) in the demethylase enzymes that are enriched in differentiation. We briefly discuss the significance of our findings within a developmental/epigenomics framework.
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