Overview of Protein Phosphorylation
Phosphorylation is the most common and important molecular mechanism of acute and reversible regulation of protein function. Through protein phosphorylation, protein function is regulated in response to extracellular stimuli both inside and outside the cell. Protein phosphorylation is usually analyzed by biosynthetic labeling with 32P-labeled inorganic phosphate (32Pi), and the studies of mammalian cells metabolically labeled with 32P orthophosphate suggest that as many as one-third of all cellular proteins are covalently modified by protein phosphorylation. Most proteins are found to be phosphorylated at serine or threonine residues, and many proteins involved in signal transduction are also phosphorylated at tyrosine residues. Lots of protein kinases exhibit a strict specificity for phosphorylation of either serine/threonine or tyrosine residues. Due to a large number of kinases and phosphatases in the genome, the identification of the specific enzymes responsible for a given site in a given protein is immensely challenging. However, because protein kinases and phosphatases recognize local specificity determinants within proteins, it is possible to use small peptides to study the characteristics of site-specific phosphorylation. In addition, phosphorylation usually causes retardation in gel mobility, providing an opportunity to investigate peptide phosphorylation and de-phosphorylation by monitoring migration on high-resolution peptide gels.
Process and Regulation of Protein Phosphorylation
Protein phosphorylation is involved in the regulation of a broad spectrum of cellular processes and states. The phosphorylation state of proteins in typical eukaryotic cells is mainly determined by the activity of protein kinases and phosphatases on their substrates. The covalent conjugation of phosphate groups to peptides frequently alters protein function by inducing conformational changes in proteins or by affecting protein-protein/enzyme-substrate interactions. Many kinases and phosphatases are also phosphorylation substrates, thereby forming mutually-dependent and hierarchically-regulated signaling loops and cascades. Most polypeptide growth factors (platelet-derived growth factor and epidermal growth factor are among the best studied) and cytokines (e.g., interleukin 2, colony stimulating factor 1, and interferon gamma) stimulate phosphorylation upon binding to their receptors.
Several signaling pathways critically involved in the embryonic development and the modulation of gene expression are initiated from the activation of cell surface growth factor receptors that are known receptor tyrosine kinases (RTKs), such as fibroblast growth factor receptor (FGFR) and insulin growth factor-1 receptor (IGF-1R), or receptor serine/threonine kinases (e.g., TGF-βR and BMPR1/2). Upon ligand binding, these receptor kinases are fully activated and phosphorylate downstream, intracellular kinases to initiate phosphorylation signaling cascades that frequently regulate the translocation and activity of several transcription factors (e.g., Myc, β-Catenin, and Smad proteins) and the expression of differentiation-related genes.
Induced phosphorylation also activates cytoplasmic protein kinases, such as Raf, the activators of the mitogen-activated protein (MAP) kinases Erk1/2 and MEK1/2, belonging to the Ras-Raf-MEK-ERK signaling pathway, and the phosphatidylinositol 3'-kinase-activated kinase (PI3K), protein kinase B/Akt. These signal transduction pathways are highly interactive with each other and inﬂuenced by other protein phosphatases including PTEN and PTPN11 (Shp2) that negatively control protein phosphorylation and also play critical roles in the modulation of this signaling network and the cell biology.
Importance of Protein Phosphorylation
Protein phosphorylation has an important role in essentially all aspects of cell biology. It was shown that the assembly of the mitotic spindle requires an accurate regulation of microtubule dynamics, which is largely achieved by cohesive work of different types of protein kinases and phosphatases. Despite the fact that serine/threonine-specific, dual and tyrosine protein phosphatases participate in the regulation of microtubule dynamics, they have different, non-overlapping functions. According to experimental data, the role of phosphatases in cytoskeleton regulation is commonly associated with serine/threonine-specific protein phosphatases: PP1, PP2A (both a catalytic and regulatory subunits), by PP4 (PPX), PP6, and PP7. Differentiation and development are also controlled by phosphorylation.
Protein de-phosphorylation also plays a vital role in the regulation of protein function. Reversible phosphorylation of proteins during the cell cycle is a key regulatory mechanism of cytoskeleton organization, dynamics, as well as in cell differentiation and division. Considerable experimental evidence now exists in favor of the role of various protein kinases as key regulators of the cytoskeleton and cell division. The number of protein kinase inhibitors with different specificity was applied in laboratory experiments and in medicine.
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