Microtubule Affinity-regulating Kinase 2 Research Articles

Abstract 3080: Novel splicing activity for RNA binding protein CUGBP2 in radiation-induced mitotic catastrophe

Abstract RNA binding protein CUGBP2 encodes various functions at the posttranscriptional level including alternative splicing (e.g. tau, troponin T), RNA editing (apolipoprotein B) and mRNA translation (cyclooxygenase-2, Mcl1). CUGBP2 contains two RNA recognition motifs (RRMs) near the N terminus, a divergent domain loaded with alanine and glutamine residues, and a third RRM near the C terminus. Based on deletion analyses, the divergent domain is believed to encode activation modules necessary for splicing activity. Recently, we have demonstrated that there are three promoters that drive the expression of the CUGBP2 gene, transcription from which results in three variant forms of the protein. Translation of the transcripts driven from the upstream promoters adds additional amino acids to the N-terminus of CUGBP2. In mouse intestine, the predominant form of the protein is variant 2. However, after exposure to 6 Gy γ-irradiation (IR), the levels of CUGBP2 mRNA shift to being predominantly variant 1. In colon cancer cells, IR induced the cells to undergo mitotic catastrophe, a poorly understood form of cell death where cells escape the G2/M checkpoint, enter mitosis prematurely and concurrently induce an apoptotic pathway. Interestingly, there was an increase in the CUGBP2 variant that is expressed from the proximal promoter (var 1). Furthermore, downregulation of CUGBP2 through specific siRNA significantly reduced the IR-induced mitotic catastrophe. While var 1 predominantly localizes to the nucleus, the other isoforms primarily reside in the cytoplasm. We next sought to determine the mechanism by which CUGBP2 induces mitotic catastrophe. We have observed that it occurs in part through inhibiting the translation of transcripts encoding anti-apoptotic proteins Bcl-2 and Mcl1. CUGBP2 also affected the splicing of two transcripts that encode microtubule-binding proteins. MARK2 (microtubule affinity regulating kinase 2) is involved in the breakdown of the microtubule network, which is important for cell division and CKAP5 (cytoskeleton associated protein 5) is a protein present in the microtubule-microfilament complex. Exons 15 and 36 are alternatively spliced in MARK2 and CKAP5, respectively. Using RT-PCR analyses, we observed that following CUGBP2 var 1 overexpression, these two exons, 15 and 36 were spliced out from the MARK2 and CKAP5 mRNA, respectively. This was associated with the cells undergoing radiation-induced mitotic catastrophe. In summary, CUGBP2 has been shown to undergo alternative promoter usage and alternative use of the first exon following exposure of cells to IR. With the increase in var 1, we observe inhibition of Bcl2 and Mcl1 translation, and increased alternative splicing of MARK2 and CKAP5. These data, taken together, suggest that CUGBP2 regulates cell viability by two different posttranscriptional mechanisms of alternative splicing and translation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3080. doi:10.1158/1538-7445.AM2011-3080

Ca2+/Calmodulin-dependent Protein Kinase IV-mediated LIM Kinase Activation Is Critical for Calcium Signal-induced Neurite Outgrowth

Actin cytoskeletal remodeling is essential for neurite outgrowth. LIM kinase 1 (LIMK1) regulates actin cytoskeletal remodeling by phosphorylating and inactivating cofilin, an actin filament-disassembling factor. In this study, we investigated the role of LIMK1 in calcium signal-induced neurite outgrowth. The calcium ionophore ionomycin induced LIMK1 activation and cofilin phosphorylation in Neuro-2a neuroblastoma cells. Knockdown of LIMK1 or expression of a kinase-dead mutant of LIMK1 suppressed ionomycin-induced cofilin phosphorylation and neurite outgrowth in Neuro-2a cells. Ionomycin-induced cofilin phosphorylation and neurite outgrowth were also blocked by KN-93, an inhibitor of Ca(2+)/calmodulin-dependent protein kinases (CaMKs), and STO-609, an inhibitor of CaMK kinase. An active form of CaMKIV but not CaMKI enhanced Thr-508 phosphorylation of LIMK1 and increased the kinase activity of LIMK1. Moreover, the active form of CaMKIV induced cofilin phosphorylation and neurite outgrowth, and a dominant negative form of CaMKIV suppressed ionomycin-induced neurite outgrowth. Taken together, our results suggest that LIMK1-mediated cofilin phosphorylation is critical for ionomycin-induced neurite outgrowth and that CaMKIV mediates ionomycin-induced LIMK1 activation.

Polarity-Regulating Kinase Partitioning-Defective 1/Microtubule Affinity-Regulating Kinase 2 Negatively Regulates Development of Dendrites on Hippocampal Neurons

Neurons are highly polarized cells that possess two morphologically and functionally different types of protrusions, axons and dendrites, that function in the transmission and reception of neural signals, respectively. A great deal of attention has been paid to the specification and guidance of axons, but the mechanism of dendrite development remains mostly unknown. We report here that a polarity-regulating kinase, partitioning-defective 1 (Par1b)/microtubule affinity-regulating kinase 2 (MARK2), specifically regulates development of dendrites in hippocampal neurons. Ectopic expression of Par1b/MARK2 shortens the length and decreases branching of dendrites without significant effects on axons. Knockdown of endogenous Par1b/MARK2 by RNA interference stimulates dendrite development. Wnt stimulation and Dishevelled expression, both of which are known to induce dendrite development, induced recruitment of Par1b/MARK2 to the membrane fraction. Expression of a Par1b/MARK2 mutant, that contains a myristoylation signal and accumulates exclusively in membranes, does not affect dendrite development. In addition, Par1b/MARK2 efficiently phosphorylated MAP2, which is localized mainly in dendrites. These results indicate that Par1b/MARK2 negatively regulates dendrite development through phosphorylation of MAP2.

A Calcium- and Calmodulin-Dependent Kinase Iα/Microtubule Affinity Regulating Kinase 2 Signaling Cascade Mediates Calcium-Dependent Neurite Outgrowth

Calcium is a critical regulator of neuronal differentiation and neurite outgrowth during development, as well as synaptic plasticity in adulthood. Calcium- and calmodulin-dependent kinase I (CaMKI) can regulate neurite outgrowth; however, the signal transduction cascades that lead to its physiological effects have not yet been elucidated. CaMKIalpha was therefore used as bait in a yeast two-hybrid assay and microtubule affinity regulating kinase 2 (MARK2)/Par-1b was identified as an interacting partner of CaMKI in three independent screens. The interaction between CaMKI and MARK2 was confirmed in vitro and in vivo by coimmunoprecipitation. CaMKI binds MARK2 within its kinase domain, but only if it is activated by calcium and calmodulin. Expression of CaMKI and MARK2 in Neuro-2A (N2a) cells and in primary hippocampal neurons promotes neurite outgrowth, an effect dependent on the catalytic activities of these enzymes. In addition, decreasing MARK2 activity blocks the ability of the calcium ionophore ionomycin to promote neurite outgrowth. Finally, CaMKI phosphorylates MARK2 on novel sites within its kinase domain. Mutation of these phosphorylation sites decreases both MARK2 kinase activity and its ability to promote neurite outgrowth. Interaction of MARK2 with CaMKI results in a novel, calcium-dependent pathway that plays an important role in neuronal differentiation.