**1. Background**

The mitogen-activated protein kinase (MAPK) pathway is one of the best described signaling system in cancer. Almost one third of all human cancers have reported alterations in MAPK signaling, indicating the high relevance of the precise understanding of this pathway [1]. The basic role of the MAPK pathway is to transduce extracellular signals into the cell to regulate fundamental cellular functions including growth, cell migration, differentiation, and apoptosis [2]. To achieve a correct regulation of these diverging functions several distinct pathways are necessary [2]. The MAPK signaling consists of three different signaling systems, the extracellular signal-regulated kinase (ERK) pathway, the C-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) pathway and the p38 kinase pathway [3]. Each of these different signaling systems is strictly hierarchically structured and consists of a MAPK kinase kinase (MAPKKK), which is superior to a MAPK kinase (MAPKK), which controls a MAPK [3]. Of these different systems, the ERK pathway is the best studied MAPK pathway. The ERK signaling can be activated by numerous extracellular stimuli, e.g., growth factors or mitogens. One example for a classical activation path is represented by the activation of the epidermal growth factor receptor (EGFR). Its hetero- or homodimerization induced by binding of epidermal growth factors leads to an autophosphorylation of the receptor [4]. Consequently, a signaling

cascade consisting of growth factor receptor-bound protein 2 (GRB2), son of sevenless (SOS), and the small GTPase Ras is activated. GTP-bound Ras recruits Raf kinases to the cell membrane for activation [4]. The Raf kinases represent the MAPKKK in the ERK pathway. Subsequently, Raf kinases activate MEK1/2 (MAPKK) and ERK1/2 (MAPK) [2]. The targets of ERK1/2 are diverse and include p90RSK, mitogen-activated protein kinase interacting protein kinases 1 and 2 (MNK1/2), Ets, Ets domain-containing protein (Elk1), Myc, signal transducer and activator of transcription 1/3 (STAT1/3) and estrogen receptor (ER), to name some of them [2]. These many targets are necessary for the precise regulation of the various aforementioned cellular functions, e.g., differentiation, growth, apoptosis, and migration.

Since, the MAPK pathway controls these essential functions a precise regulation and titration of the signaling activity is needed. A perfect example for such a strict regulation is the upstream positioned Raf kinases. The Raf kinase family consists of three different Raf proteins, Raf-1, B-Raf and A-Raf. Although their structures are almost similar, their activation modes are extremely different. After recruitment of Raf kinases to GTP-bound Ras, a complex series of phosphorylations is induced for activation. These phosphorylations are needed for the activation of the kinase domain and reduction of the autoinhibition. The activation of Raf-1 and A-Raf requires phosphorylation of the N-region, dephosphorylation of the S259 inhibitory site, and phosphorylation of the activation loop. B-Raf is already in a preactivated state and can be fully activated by Ras alone, whereas the activation of Raf-1 and A-Raf requires other factors [1]. Due to this, only small aberrations in the structure of B-Raf, such as the V600E exchange, are needed to induce a constitutive activation. Alterations such as these are found in almost two thirds of malignant melanoma and in glioblastoma, where of more than 40 different mutations in the B-Raf gene 90% are at residue 600 in exon 15 [5].

Another regulating mode of the Raf kinases is the binding to scaffolding proteins such as the 14-3-3 protein family [6]. Such adaptor and scaffolding proteins facilitate the correct subcellular localization, provide a proximity of different signaling partners and support the formation of multiprotein complexes [3]. Moreover, scaffolding proteins can shield activated signaling molecules from deactivating phosphatases to allow an adequate signaling strength [3]. Additionally, scaffolds provide crosstalks between different signaling pathways.

Of note, the protein tyrosine phosphatase interacting protein 51 (PTPIP51) represents another scaffold protein, which regulates MAPK activation on Raf-1 level [7]. PTPIP51 exerts its regulating effect on the MAPK pathway on Raf-1 level via the scaffold protein 14-3-3β [7]. The recruitment of PTPIP51 into the MAPK signaling leads to an activation of the MAPK pathway. A well-titrated signal is a prerequisite for an optimal cellular function. Therefore, the formation of the PTPIP51/14-3-3β/Raf-1 complex is tightly regulated by kinases and phosphatases [8,9]. One of the crucial spots for this regulation is the tyrosine 176 residue of PTPIP51 [9–11]. Its phosphorylation results in a break-up of the PTPIP51/14-3-3β/Raf-1 complex and hence the stimulation of the MAPK signaling is omitted [8,9,11,12]. The phosphorylation of the tyrosine 176 residue is under the control of the EGFR and other kinases, such as the cellular sarcoma kinase (c-Src) [8,10–12]. Dephosphorylation is mainly performed by PTP1B [9,11,12]. Another important phosphorylation site of PTPIP51 is the serine 212 residue. Computational models of the PTPIP51 molecule show a cleft in its tertiary structure, which is surrounded by the aforementioned tyrosine 176 residue and serine 212 residue, respectively [9]. Up to now, we assume, that the cleft represents a binding site for the Raf kinases [9]. Contrary to the interaction inhibiting tyrosine 176 residue, phosphorylation of the serine 212 residue leads to an augmentation of the interaction with Raf-1 via 14-3-3β [7–9,12]. Besides the cleft, PTPIP51 protein structure contains tetratricopeptide domains, which are known to serve as binding sites for protein-protein interactions [9]. Additionally, in the structure of PTPIP51 two conserved regions are found. These sites facilitate the interaction with the scaffolding protein 14-3-3β [7,9]. In summary, PTPIP51 possesses the perfect scaffolding protein equipment, encompassing several binding sites for protein-protein interactions and the capability of modulating these bindings via phosphorylation and dephosphorylation of tyrosine and serine residues (Figure 1A).

Besides the direct regulation of the MAPK pathway, PTPIP51 is involved in a broad range of cellular functions and signaling systems. The panel of interaction partners ranges from NF-κB signaling proteins (RelA, I-κB) over mitochondrial associated ER membrane-related proteins (VAPB, ORP5/ORP8), autophagy-related signaling, and mitosis associated proteins (CGI-99, Nuf2) [13–20]. These interactions of PTPIP51 are already reviewed and analyzed by studies of our group and other scientists. Therefore, the focus of this review is to highlight the regulation of PTPIP51 and its functional consequences affecting the MAPK signaling in diseases associated with an aberrant MAPK signaling.

**Figure 1.** Regulation of PTPIP51 interactions in normal cells (represented by the HaCat cell line). Activation of the EGFR via the binding of EGF induces an activation of Raf-1 via several signaling molecules. Raf-1 depicts the MAPKKK of the ERK signaling. Its activation triggers a signaling cascade via MEK1/2 and ERK1/2, which ultimately initiates transcription. The EGFR also phosphorylates the Tyr176 residue of PTPIP51 and thereby inhibits its interaction with Raf-1. This mechanism prevents an overshooting activation of the MAPK pathway. The right side of the figure represents the interactions when EGFR is inhibited. The inhibition of EGFR leads to an omission of Tyr176 phosphorylation of PTPIP51 via the EGFR. The dephosphorylation of PTPIP51 at Tyr176 induces the formation of the Raf-1/14-3-3β/PTPIP51 complex and thus a stimulation of the MAPK pathway. This mechanism partially compensates for the EGFR inhibition (black arrows indicate a phosphorylation/activation; dotted black arrows indicate a reduced phosphorylation/activation) (**A**); regulation of PTPIP51 interactions in insulin resistance. Activation of the insulin receptor induces the activation of the PI3K-Akt-mTOR signaling and the MAPK pathway, especially the ERK signaling. Here, PTPIP51 stimulates the signaling on Raf-1 level and potentially modulates the insulin sensitivity on transcriptional level. Protein kinase A (PKA) phosphorylates the Ser46 residue of PTPIP51 and thereby stimulates the binding of PTPIP51 and Raf-1 via 14-3-3β (black arrows indicate a phosphorylation/activation) (**B**).
