Protein Kinase Activity and ATP Binding

Protein Kinases are enzymes that catalyze the phosphorylation of proteins as post-translational modifications. The addition of the phosphate groups helps to regulate many cellular processes and causes a conformational change for the phosphorylated protein. Typically, the phosphorylation of a protein makes it more active/sends a signal further down a signal pathway. (17)

Phosphorylation of eukaryotic proteins only occurs at serine, threonine, or tyrosine residues. All of these residues have nucleophilic hydroxyl groups that attack the gamma phosphate group of ATP, causing a transfer of the phosphate groups. (17)

About 80% of mammalian protein kinase enzymes are serine/theronine kinases, like Braf. These kinases are specific to their substrates through conserved polypeptide binding regions in their structure. ATP is the most common source of phosphate groups for phosphorylation, and the breaking off of one of ATP's phosphate groups releases free energy and causes unidirectionality of the phosphorylation process. (17)

ATP binding sites are conserved across the various kinases. Raf kinases have a conserved structure of a small N-terminal lobe and large C-terminal lobe. The smaller lobe binds and conforms ATP for use via its glycine rich P-loop (ATP phosphate binding loop). The site of phosphorylation catalysis is between the Nt and Ct lobes and can open/close relative to each other allowing for the switching between its active/inactive state. (27)

Braf's catalytic site is represented by a K/D/D motif. Lysine is conserved through kinases as the site of ATP binding. At Bra'f K578 residue, a salt bridge forms between the kinase and an ATP molecule, and at D578 a serine or threonine residue from another protein is oriented to catalyze the nucleophilic attack facilitated phosphate transfer. (27)

Braf and the MAPK Cascade

The general reaction that Braf catalyzes is ATP+Protein=ADP+Phosphoprotein

Meaning that Braf primarily functions as a kinase, phosphorylating its substrates. (26)

MAPK signaling pathways are used throughout the body in order to regulate different cell processes. MAPK pathways control cell growth, differentiation and apoptosis. MAPK signaling cascades initially begin with an external stimuli that is then sent through the cell through a series of kinase reactions. (33)

Braf is involved in the ERK 1/2 MAPK pathway. This pathways begins with the binding of the signaling ligand to its receptor and the signal is sent to GTPase Ras. GTPase Ras activates Raf-1, Braf and other Raf proteins at their RAS binding domains (http://www.sciencedirect.com/science/article/pii/S0092867404002156). It is thought that B-raf and C-Raf dimerize to become active. B-raf phosphorylates 2 serine residues of MEK 1/2. MEK 1/2 then continues the signaling cascade by phosphorylating ERK 1/2. ERK 1/2 can then go on to phosphorylate substrates either in the cytosol, nucleus, or other organelles. (33)

Therefore, it is clear that through its role in the MAPK signaling cascade pathway, Braf is integral in cell processes involving cell growth or death and the signaling of other proteins that more directly control gene expression.

Among the numerous biological processes that MAPK Signaling Pathway (and therefore Braf functioning) is apart of is Focal Adhesion , Neurotrophin Signaling Pathway , and the Insulin Signaling Pathway (7)

Vascular Smooth Muscle Contraction and Braf

In the pathway stimulating vascular smooth muscle contraction, Braf is an integral part of the signaling pathway. (7) Protein Kinase C (PKC) phosphorylates Raf, which phosphorylates MEK. MEK phosphorylates ERK, ERK phosphorylates CaD (Caldesmon, which is a strongly binds actin and is thought to be a regulatory point for muscle contraction. CaD is regulated via Calcium binding and phosphorylation. (32)

Sub-cellular localization of Braf

Braf is localized in the nucleus, cytoplasm and cell membrane of the cell. (7) This makes sense considering its involvement in the MAPK signaling cascade that controls cell growth and other processes that involves signals being sent from the cell membrane to the nucleus in order to regulate transcription.

Additionally, Braf has been found in the mitochondria and other subcellular organelles. (7)