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1.6.1 MAPK Family Members and Their Biological Functions

The mitogen-activated protein kinase (MAPK) family is an important kinase family in mammalian cells, consisting of four subfamilies: extracellular-regulated protein kinase1 and 2 (ERK1/2), ERK5, c-jun NH2-terminal protein kinases (JNK), and p38 MAPKs (Graves, Campbell et al. 1995; Seger and Krebs 1995; Attar, Atten et al. 1996; Hu and Plaxton 1996). The biological functions of MAPK pathways are diverse, playing roles in many cellular processes. In general, ERK subfamilies are related to cell growth, proliferation and survival, induced by growth factor stimulations. JNK and p38 MAPKs are associated with inflammation, apoptosis, cell growth and differentiation induced by cytokine stimulation, as well as extracellular stresses (Obata, Brown et al. 2000;

Hardwick, van den Brink et al. 2001; Papatsoris and Papavassiliou 2001; Saldeen, Lee et al. 2001).

All the MAPK family members are activated by phosphorylation at Threonine and Tyrosine residues within the activation loop TxY of kinase subdomain VIII by diverse activators: mitogen-activated protein kinase kinase (MEK1) and MEK2 for ERK1/2, MEK5 for ERK5, MKK4 and MKK7 for JNKs, MKK3 and MKK6 for p38 MAPKs (Graves, Campbell et al. 1995). The x in the TxY motif of the kinase subdomain VIII is diverse in MAPKs. For example, x is proline in JNK, glutamate in ERK, glycine in p38 MAP kinases. MEKs are activated by an upstream activator, MEK kinase. There are many MEK kinases that activate MEKs: Raf-1, A-Raf, B-Raf, c-Raf, Mos, Tpl2 are for MEK1 and MEK2; MEKK1, MEKK2, MEKK3, Tpl2 are for MEK5; PAK is for MKK3 and MKK6; MEKK4 and DLK are for MKK4 and MKK7. Cross-talk is always occurring among these MAPK signaling pathways. In addition, some activators are involved in more than one MAPK pathway. For example: the activators for MEK5 can also activate MEK1 and MEK2. In general, MAPK cascade members use docking interactions to regulate the efficiency and specificity between upstream activators and downstream substrates (Hu, Shen et al. 2007). In MAP2ks, such as MKK6, the docking domains are located at the N-terminal or C-terminal to bind its activator, MAP3Ks. The docking domain of MEK5, consisting of amino acids sequence, EYEDEDGD, is located in its N- terminus which interacts with its upstream regulator, MEKK2 or its downstream substrate, ERK5 (Seyfried, Wang et al. 2005). For MAPKs, such as ERK2, the docking domain is located in both the N- and C-terminals. ERK2 has a CD domain (common domain) consisting of a group of negatively charged amino acids, which is located in the

C-terminal region just after its kinase domain. ERK2 also has a docking domain named the ED site, which is located at its N-terminal region in front of its kinase domain. MAPK substrates have docking domains for their regulation by MAPK. C-Jun has a D domain to interact with its activator, JNK. c-Fos has DEF (docking site for ERK or FX) domain in its C-terminal region to interact with ERK. Some ERK substrates have both D domain and DEF domain to interact with their activators, such as JNK and ERK. In addition, some MAPK cascades use scaffold proteins to assemble functional MAPK modules to increase the interaction specificity. For example, JIP-1, a scaffold protein in the JNK pathway, contains a D domain to bind JNK (Dickens, Rogers et al. 1997)

On the other hand, the activation of MAPK is negatively controlled by MAPK phosphatase activity. Any dephosphorylation at either Threonine or Tyrosine residues in TxY motif inactivates MAPK. The dephosphorylation and inactivation process in the activation loop in MAPK last from minutes to hours depending on cell type and stimuli. Serine/Threonine protein phosphotases, such as PP2A, a member from the PPP family, inactivates ERKs. Members of Tyrosine protein phosphotase, such as PTP-SL, STEP, and HePTP, also dephosphorylate and inactivate ERKs. MAPK phosphatase plays important roles in diverse biological functions. For example, the JNK phosphatase family, the puckered mutation leads to cytoskeletal defects resulting in dorsal closure during Drosophola embryogensis. Interestingly, a subclass of the tyrosine phosphotase family called dual specificity phosphatase (DSP) dephosphorylate both tyrosine and threonine residues to inactivate MAPKs (Camps, Nichols et al. 2000). There are nine members identified in DSP family, such as CL100/MKP-1, PAC1, hVH-2/MKP-2, hVH3/B23, hVH-5, MKP-3/PYST1, B59, and MKP-5. PAC1, hVH-2/MKP-2,

hVH3/B23, hVH-5 are expressed exclusively in the nucleus and MKP-3/PYST1 is expressed in the cytosol. MKP-4 is expressed in both the cytosol and the nucleus.

1.6.2 ERK5-MEK5 Signal Pathway

Extracellular signal-regulated kinase 5 (ERK5), which is also named big-MAPK or BMK1, is one of the important MAPK family members, and it is activated through a cascade of signal transduction by MAP3Ks (MEKK2/3 and Cot), MEK5 and ERK5. ERK5 activation is exclusively activated by MEK5. The MEK5 signal pathway was first discovered in 1995 by Dixon and co-workers with a yeast two-hybrid screen experiment. ERK5 was used as the bait to interact with MEK5. ERK5-MEK5 signal pathway plays important roles in cell growth, cell differentiation, neuronal survival and embryonic angiogenesis (Sohn, Sarvis et al. 2002; Wang, Merritt et al. 2005). There are many transcription factors that can be phosphorylated by ERK5, such as c-Myc, MEF2 family members and c-Fos (Kamakura, Moriguchi et al. 1999) (Yang, Ornatsky et al. 1998). ERK5 has two splicing variants, ERK5 and ERK5b. ERK5b is shorter in the N-terminal compared with ERK5. ERK5b is a naturally dominant negative mutant which regulates ERK5 kinase activity. Overexpression of ERK5b inhibits ERK5b kinase function on nuclear substrate, such as MEF2C.

The MEK5 gene codes a 444 amino acids protein, which has two splicing variants, MEK5a and MEK5b (English, Vanderbilt et al. 1995; Chao, Hayashi et al. 1999). MEK5a is a protein with 89 amino acids longer than MEK5b at the N-terminal.

Both of MEK5a and MEK5b can individually interact with ERK5. Double phosphorylated MEK5a at Serine 311 and Threonine 315 activates ERK5 as well as its

nuclear translocation, but not MEK5b (Kato et al., 1997). MEK5a directly interacts and stimulates ERK5 kinase activity by phosphorylating threonine and Tyrosine residues in the TxY motif. On the other hand the overexpression of MEK5b disrupts the interaction of MEK5a with ERK5, which indicates MEK5b is a putative dominant negative mutant which regulates MEK5a kinase function to activate ERK5.

The expression and cellular localization of MEK5a and MEK5b are tissue specific (English, Vanderbilt et al. 1995). MEK5a is localized dominantly in the cytosol and MEK5b is distributed evenly in the whole cell. MEK5a expression is abundant in actively mitotic tissues, such as liver and brain (English, Vanderbilt et al. 1995). MEK5b has more expression than MEK5a in differentiated tissues, such as muscle, lung, kidney et al. Many studies showed the expression of MEK5 is closely related with tumorigenesis. MEK5 is overexpressed in human prostate cancer cells as detected by antibodies that recognize the specific N-terminal of MEK5a (Mehta, Jenkins et al. 2003). Immunohistostaining results also show that MEK5 is overexpressed in human prostate cancer but not in benign prostate tissues (Mehta, Jenkins et al. 2003). Moreover, the expression level of MEK5 in malignant prostate cancer is correlated with its bone metastases (Mehta, Jenkins et al. 2003). Patients with higher expression of MEK5 had a shorter disease-specific survival time (52 months) compared to those with weaker MEK5 expression which survived in 58 months. The activation of MEK5 induces prostate cancer cell proliferation. The overexpression of a constitutively active MEK5 mutant (MEK5D) in LNCaP cells, a metastatic prostatic adenocarcinoma cell line derived from the lymph node, induces a 3.7 fold higher cell proliferation compared to control vectors. In our experiments the expression of MEK5 is upregulated by PKM2 overexpression,

which results in cell proliferation, especially in more metastatic cancer cell lines compared to the less aggressive cell lines. Moreover, MEK5D overexpression in LNCaP cells increases cell migration by 1.8 fold and invasion ability by 2.1 fold compared to control vectors, which indicates that MEK5 plays a role in tumor metastasis as well as invasion. MEK5 is a putative modulator in regulating gene expression involved in tumor metastasis and migration processes. Matrix metalloproteinases 9 (MMP9), a zinc- dependent enzyme involved in the metastasis process, is transcriptionally upregulated by MEK5 overexpression (Mehta, Jenkins et al. 2003).