INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 40: 271-280, 2017 The crucial role of protein phosphorylation in cell signaling and its use as targeted therapy (Review) FATIMA ARDITO, MICHELE GIULIANI, DONATELLA PERRONE, GIUSEPPE TROIANO and LORENZO LO MUZIO Department of Clinical and Experimental Medicine, Foggia University, I-71122 Foggia, Italy Received January 17, 2017; Accepted May 11, 2017 DOI: 10.3892/ijmm.2017.3036 Abstract. Protein phosphorylation is an impo rtant inhibitors can be valuable for the treatment of cancer. In this cellular regulatory mechanism as many enzymes and review, we discuss the mechanism of action of phosphorylation, receptors are activated/deactivated by phosphorylation and with particular attention to the importance of phosphorylation dephosphorylation events, by means of kinases and phosph- under physiological and pathological conditions. We also atases. In particular, the protein kinases are responsible discuss the possibility of using kinase inhibitors in the for cellular transduction signaling and their hyperactivity, treatment of tumors. malfunction or overexpression can be found in several diseases, mostly tumors. Therefore, it is evident that the use of kinase Contents 1. Introduction Correspondence to: Professor Lorenzo Lo Muzio, Department 2. Protein kinases of Clinical and Experimental Medicine, Foggia University, 3. Protein phosphatases Via Rovelli 48, I-71122 Foggia, Italy 4. Activities and role of protein phosphorylation under E-mail: [email protected] physiological conditions 5. Protein phosphorylation and cancer Abbreviations: ADP, adenosine diphosphate; AGC kinases, 6. Protein kinases as drug targets protein kinase A, G and C families; Asp or D, aspartate residues; ATP, adenosine triphosphate; CaMK, Ca2+/calmodulin-dependent 7. Conclusions protein kinase class; cAMP/cGMP, cyclic-adenosine monopho- sphate/cyclic-guanosine monophosphate; Cdc25, cell division cycle 25 phosphatase; CK1, casein kinase 1; CML, chronic myeloid 1. Introduction leukemia; c-Src, proto-oncogene tyrosine-protein kinase; DAP protein, death-associated protein; DRAK2, apoptosis-inducing Protein phosphorylation is one of the most common and kinase 2; DSKs, dual-specificity kinases; EGF, epidermal growth important post-translational modifications (PTMs) (1,2). This factor; GISTs, gatrointestinal stromal tumors; Grb2, growth factor reversible mechanism occurs through protein kinases and receptor-bound protein 2; HGF, hepatocyte growth factor; HIF-1α, consists of the addition of a phosphate group (PO4) to the polar hypoxia-inducible factor-1α; His or H, histidine residues and group R of various amino acids. Consequently, this addition N-phosphorylation; IFN-γ, interferon-γ; IRS-1, insulin receptor modifies the protein from hydrophobic apolar to hydrophilic substrate-1; mRNA, messenger ribonucleic acid; Neph1, glomerular podocyte protein nephrin 1; PDGFRs, platelet-derived growth factor polar, allowing the protein to change conformation when interacting with other molecules. A phosphorylated amino receptors; PO4, phosphate group; PP1, phosphoprotein phosphatase 1; PP2A, phosphoprotein phosphatase 2A; PP2B, phosphoprotein acid can bind molecules able to interact with other proteins phosphatase 2B; PP2C, phosphoprotein phosphatase 2C; and consequently assemble and detach proteic complexes (3). PP4-7 phosphoprotein phosphatase 4-7; PPM, metallo-dependent The interactive capacity of the phosphate group is mainly protein phosphatase family; PPP, phosphoprotein phosphatase due to its components. One of its main elements is phosphorus. family; pSer/pThr, phosphoserine and phosphothreonine; PTMs, It has five outer electrons able to form a maximum of five post-translational modifications; PTP, protein-tyrosine phosphatase covalent bonds, has three pKas, high water solubility and it can family; pTyr, phosphotyrosine; Ser or S, serine residues; Ser350, form, for its versatility, mono, di and trialkyl and aryl esters serine/threonine-protein kinase; SNAP25, synaptosomal-associated with hydroxyl groups, but also acid anhydrides (4). protein 25; STKs, serine/threonine kinases; Thr or T, threonine In particular, many cellular phosphate esters are phos- residues; TKs, tyrosine kinases; TKLs, tyrosine kinase-like kinases; Tyr or Y, tyrosine residues; VEGFRs, vascular endothelial growth phoproteins that form, via a catalytic enzyme and adenosine factor receptors triphosphate (ATP), a phosphate anhydride, acting as a donor of a phosphate group. Key words: protein phosphorylation, kinase, phosphatase, A good energy balance also favors phosphorylation. phospho-signaling networks, cancer, drug target Indeed, there is a constant balance between phosphorylation and dephosphorylation events mediated by kinases, 272 ARDITO et al: PROCESS OF PROTEIN PHOSPHORYLATION AND TARGETED THERAPY phosphatases, ATP and/or ADP (protein + ATP ⇄ phosph- phorylation of the activation loop or through an allosteric oprotein + ADP) (5,6) (Fig. 1). mechanism (8). Moreover, the kinases also have non-catalytic The Cell Signaling Technology PhosphoSitePlus (www. domains allowing the attachment of substrates and the recruit- phosphosite.org) and the Kinexus PhosphoNET (www.phos- ment of other signaling proteins (9). phonet.ca) websites both list over 200,000 known human Up to 30% of all human proteins may be modified by phosphosites, and the Kinexus website predicts another 760,000 kinase activity, and kinases are known to regulate the majority additional sites that are likely to be phosphorylated. More of cellular pathways, especially those involved in signal trans- than two-thirds of the 21,000 proteins encoded by the human duction (10). genome has been shown to be phosphorylated, and it is likely In the last few years, kinases have been considered impor- that more than 90% are actually subjected to this type of PTM. tant not only for their crucial role in signaling but also for the More than one-third of the protein phosphorylation events transduction of the signal, controlling its amplitude (11-13). To occurs on serine (Ser or S), threonine (Thr or T), and tyrosine facilitate the study of phospho-signaling networks, different residues (Tyr or Y) (O-phosphorylation) (7). In particular, databases have been designed (2,14,15). the phosphorylated residues of serine are 86.4%, followed by The 518 human protein kinases are classified according to residues of threonine 11.8% whereas only 1.8% of tyrosine the amino acid residue that it phosphorylates. Most kinases residues are phosphorylated (8,9). Tyrosine phosphorylation is act on both serine and threonine (serine/threonine kinases; relatively rare compared to the other PTMs and is typical of STKs), others act on tyrosine (tyrosine kinases; TKs), and a the epidermal growth factor receptor (EGFR) family, which number act on all three (dual-specificity kinases; DSKs) (16). owns a domain called, precisely, tyrosine kinase. Sometimes, The latter can phosphorylate STKs and TKs (17); at least 125 phosphorylation of histidine (His or H) and aspartate resi- of the human protein kinases are STKs (18). dues (Asp or D) (N-phosphorylation) also occurs, but, in both The STKs are enzymes that phosphorylate the OH group cases, this phosphorylation is less stable than others. of serine or threonine, and are activated by different events Protein phosphorylation is a mechanism of regulation such as DNA damage or chemical signals mediated for that is extremely important in most cellular processes such as instance by Ca2+/calmodulin, cyclic-adenosine monopho- protein synthesis, cell division, signal transduction, cell growth, sphate/cyclic-guanosine monophosphate (cAMP/cGMP) and development and aging as many enzymes and receptors are diacylglycerol. activated and deactivated via phosphorylation/dephosphoryla- There are also the following subfamilies of protein tion events due to specific kinases and phosphatases (10). kinases: AGC, CaMK, CK1, CMGC, STE, TK and The human genome, in fact, includes approximately TKL (19-27) (Table I). 568 protein kinases and 156 protein phosphatases that regulate phosphorylation events and, therefore, play an important role 3. Protein phosphatases in the control of biological processes such as proliferation, differentiation and apoptosis. Phosphatases have the opposite function of kinases. They For instance, p53 protein is activated by phosphorylation remove the phosphate group from phosphoproteins by hydro- and is then able to stimulate transcription of genes to inhibit lyzing phosphoric acid monoesters into a phosphate group and the cell cycle, activate DNA repair and in some cases lead a molecule with a free hydroxyl group (28,29). to apoptosis (13). An imbalance in the mechanism of phos- Enzymatic removal reverts the protein to a non-phosphory- phorylation/dephosphorylation of the p53 protein can lead to lated state with a kinetics more rapid than kinases (30). When a chronic inactivation of the protein itself, which in turn can working with proteins in the laboratory, phosphatases are transform the cell into a cancer cell. inactivated using denaturation or inhibitors so phosphoryla- tion inside of a sample is not lost (31). 2. Protein kinases The protein phosphatases are considered passive house- keeping enzymes compared with protein kinases; their The protein kinases belong to the great family of kinases and different structure makes them harder to identify and less are responsible for the mechanism of phosphorylation. They important than the protein kinases (32). are activated by phosphorylation