Search

Activation of the Ras-MAPK signal transduction pathway is necessary for biological responses both to growth factors and ECM. Here, we provide evidence that phosphorylation of S298 of MAPK kinase 1 (MEK1) by p21-activated kinase (PAK) is a site of convergence for integrin and growth factor signaling. We find that adhesion to fibronectin induces PAKl-dependent phosphorylation of MEK1 on S298 and that this phosphorylation is necessary for efficient activation of MEK1 and subsequent MAPK activation. The rapid and efficient activation of MEK and phosphorylation on S298 induced by cell adhesion to fibronectin is influenced by FAK and Src signaling and is paralleled by localization of phospho-S298 MEK1 and phospho-MAPK staining in peripheral membrane-proximal adhesion structures. We propose that FAK/Src-dependent, PAK1-mediated phosphorylation of MEK1 on S298 is central to the organization and localization of active Raf-MEK1-MAPK signaling complexes, and that formation of such complexes contributes to the adhesion dependence of growth factor signaling to MAPK.

Indirect immunofluorescence was used to study possible involvement of mitogen-activate protein (MAP) kinases ERK1 and ERK2 in somatic cell mitosis. Part of the active MAP kinase is localized at kinetochores, asters and the mid-body during mitosis. The kinetochore motor protein CENP-E was identified as a candidate mitotic substrate for MAP kinase. MAP kinase is proposed to play a part in mitosis by changing the ability of CENP-E to mediate chromosome and microtubule interactions.

Vascular endothelial growth factor receptor inhibitors (VEGFRis) improve cancer survival but are associated with treatment-limiting hypertension, often attributed to endothelial cell (EC) dysfunction. Using phosphoproteomic profiling of VEGFRi-treated ECs, drugs were screened for mitigators of VEGFRi-induced EC dysfunction and validated in primary aortic ECs, mice, and canine cancer patients. VEGFRi treatment significantly raised systolic blood pressure (SBP) and increased markers of endothelial and renal dysfunction in mice and canine cancer patients. α-Adrenergic-antagonists were identified as drugs that most oppose the VEGFRi proteomic signature. Doxazosin, one such α-antagonist, prevented EC dysfunction in murine, canine, and human aortic ECs. In mice with sorafenib-induced-hypertension, doxazosin mitigated EC dysfunction but not hypertension or glomerular endotheliosis, while lisinopril mitigated hypertension and glomerular endotheliosis without impacting EC function. Hence, reversing EC dysfunction was insufficient to mitigate VEGFRi-induced-hypertension in this mouse model. Canine cancer patients with VEGFRi-induced-hypertension were randomized to doxazosin or lisinopril and both agents significantly decreased SBP. The canine clinical trial supports safety and efficacy of doxazosin and lisinopril as antihypertensives for VEGFRi-induced-hypertension and the potential of trials in canines with spontaneous cancer to accelerate translation. The overall findings demonstrate the utility of phosphoproteomics to identify EC-protective agents to mitigate cardio-oncology side effects., (© 2024 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

INTRODUCTIONThis protocol describes a method for detection of direct substrates of a protein kinase in cell lysates or fractions. The approach involves the addition of recombinant mutant kinase and [γ-(32)P]ATP analog to cell lysates. This technique has been successfully used for the phosphorylation of extracellular signal-regulated kinase 2 (ERK2) substrates in cell lysates from SKOV-3 ovarian cells; however, the methodology can be applied to other protein kinases as well.

INTRODUCTIONThe generation of a high-specific-activity [γ-(32)P]ATP analog is essential for detecting direct substrates of a kinase. Synthesis of radiolabeled ATP analog from unlabeled ADP analog involves a succession of phosphotransfer reactions that are performed in two stages. The first stage involves transfer of phosphate from [γ-(32)P]ATP to nucleotide diphosphate kinase (NDPK) to make [(32)P]NDPK. The second stage involves transfer of labeled phosphate from [(32)P]NDPK to ADP analog to generate [γ-(32)P]ATP analog. In this protocol, radiolabeled [γ-(32)P]cpATP is prepared from cyclopentyl ADP (cpADP) and [γ-(32)P]ATP by a phosphate transfer reaction using NDPK.

INTRODUCTIONThis protocol describes a method for detection of direct substrates of a protein kinase in cell lysates or fractions. The approach involves identification of kinase-associated substrates by immunoprecipitating a tagged form of the mutant kinase from transfected COS-1 cells and performing a kinase reaction by the addition of [γ-(32)P]ATP analog. This technique has been used for the phosphorylation of extracellular signal-regulated kinase 2 (ERK2) substrates; however, the methodology can be applied to other protein kinases as well.

INTRODUCTIONThis protocol describes a method for studying protein kinases and their substrates by generation of mutant kinase enzymes that can incorporate ATP analogs into the substrate. This method was used to identify substrates of extracellular signal-regulated kinase 2 (ERK2). Once mutations (called "pocket mutations") have been generated in the kinase, the next step is to screen ATP analogs for their compatibility with the kinase mutant. This screening is best performed in a two-step process. The first step involves assaying the ability of an ATP analog to inhibit the incorporation of radioactive phosphate from normal [γ-32P]ATP into a known substrate in an in vitro kinase reaction. Because certain analogs might be able to interact with the ATP-binding site of the kinase but be unable to be used by the kinase as an ATP source, it is also necessary to directly test the ability of the mutant kinase to phosphorylate a substrate with analog ATP. Doing this requires either radiolabeled ATP analogs or, preferably, a phospho-specific antibody to the phosphorylation site on the known substrate. (A phospho-specific antibody allows a large number of ATP analogs to be screened without the need for radioactivity.) The kinase/ATP analog pair that provides the best phosphorylation of the substrate is then used for future experiments. Only those analogs that cannot be used by the wild-type kinase and other cellular kinases are chosen.

Specificity in signal transduction can be achieved through scaffolds, anchors, and adapters that assemble generic signal transduction components in specific combinations and locations. MEK Partner-1 (MP1) was identified as a potential "scaffold" protein for the mammalian extracellular signal-regulated kinase (ERK) pathway. To gain insight into the interactions of MP1 with the ERK pathway, we analyzed the ability of MP1 to bind to MEK1, ERK1, and to itself, and the regulation of these interactions. Gel filtration of cell lysates revealed two major MP1 peaks: a broad high molecular weight peak and a 28 kDa complex. An MP1 mutant that lost MEK1 binding no longer enhanced RasV12-stimulated ERK1 activity, and functioned as a dominant negative, consistent with the concept that MP1 function depends on facilitating these oligomerizations. Activation of the ERK pathway by serum or by RasV12 did not detectably affect MP1-MP1 dimerization or MP1-MEK1 interactions, but caused the dissociation of the MP1-ERK1 complex. Surprisingly, pharmacological inhibition of ERK activation did not restore the complex, suggesting that regulation of complex formation occurs independently of ERK phosphorylation. These results support the concept that MP1 functions as a regulator of MAP kinase signaling by binding to MEK1 and regulating its association with a larger signaling complex that may sequentially service multiple molecules of ERK.