Yoav D. Shaul

ERK1c regulates Golgi fragmentation during mitosis

Extracellular signal-regulated kinase 1c (ERK1c) is an alternatively spliced form of ERK1 that is regulated differently than other ERK isoforms. We studied the Golgi functions of ERK1c and found that it plays a role in MEK-induced mitotic Golgi fragmentation. Thus, in late G2 and mitosis of synchronized cells, the expression and activity of ERK1c was increased and it colocalized mainly with Golgi markers. Small interfering RNA of ERK1c significantly attenuated, whereas ERK1c overexpression facilitated, mitotic Golgi fragmentation. These effects were also reflected in mitotic progression, indicating that ERK1c is involved in cell cycle regulation via modulation of Golgi fragmentation. Although ERK1 was activated in mitosis as well, it could not replace ERK1c in regulating Golgi fragmentation. Therefore, MEKs regulate mitosis via all three ERK isoforms, where ERK1c acts specifically in the Golgi, whereas ERK1 and 2 regulate other mitosis-related processes. Thus, ERK1c extends the specificity of the Ras-MEK cascade by activating ERK1/2-independent processes.

Targeting of PYK2 to focal adhesions as a cellular mechanism for convergence between integrins and G protein-coupled receptor signaling cascades.

The non-receptor tyrosine kinase PYK2 appears to function at a point of convergence of integrins and certain G protein-coupled receptor (GPCR) signaling cascades. In this study, we provide evidence that translocation of PYK2 to focal adhesions is triggered both by cell adhesion to extracellular matrix proteins and by activation of the histamine GPCR. By using different mutants of PYK2 as green fluorescent fusion proteins, we show that the translocation of PYK2 to focal adhesions is not dependent on its catalytic activity but rather is mediated by its carboxyl-terminal domain. Translocation of PYK2 to focal adhesions was attributed to enhanced tyrosine phosphorylation of PYK2 and its association with the focal adhesion proteins paxillin and p130Cas. Translocation of PYK2 to focal adhesions, as well as its tyrosine phosphorylation in response to histamine treatment, was abolished in the presence of protein kinase C inhibitors or cytochalasin D treatment, whereas activation of protein kinase C by phorbol ester resulted in focal adhesion targeting of PYK2 and its tyrosine phosphorylation in an integrin-clustering dependent manner. Overexpression of a wild-type PYK2 enhanced ERK activation in response to histamine, whereas a kinase-deficient mutant substantially inhibited this response. Furthermore, inhibition of PYK2 translocation to focal adhesions abolished ERK activation in response to histamine treatment. These results suggest that PYK2 apparently links between GPCRs and focal adhesion-dependent ERK activation and can provide the molecular basis underlying PYK2 function at a point of convergence between signaling pathways triggered by extracellular matrix proteins and certain GPCR agonists.

Extracellular Signal-Regulated Kinase 1c (ERK1c), a Novel 42-Kilodalton ERK, Demonstrates Unique Modes of Regulation, Localization, and Function

ABSTRACT Extracellular signal-regulated kinases (ERKs) are signaling molecules that regulate many cellular processes. We have previously identified an alternatively spliced 46-kDa form of ERK1 that is expressed in rats and mice and named ERK1b. Here we report that the same splicing event in humans and monkeys causes, due to sequence differences in the inserted introns, the production of an ERK isoform that migrates together with the 42-kDa ERK2. Because of the differences of this isoform from ERK1b, we named it ERK1c. We found that its expression levels are about 10% of ERK1. ERK1c seems to be expressed in a wide variety of tissues and cells. Its activation by MEKs and inactivation by phosphatases are slower than those of ERK1, which is probably the reason for its differential regulation in response to extracellular stimuli. Unlike ERK1, ERK1c undergoes monoubiquitination, which is increased with elevated cell density concomitantly with accumulation of ERK1c in the Golgi apparatus. Elevated cell density also causes enhanced Golgi fragmentation, which is facilitated by overexpression of native ERK1c and is prevented by dominant-negative ERK1c, indicating that ERK1c mediates cell density-induced Golgi fragmentation. The differential regulation of ERK1c extends the signaling specificity of MEKs after stimulation by various extracellular stimuli.

Targeting of Nir2 to Lipid Droplets Is Regulated by a Specific Threonine Residue within Its PI-Transfer Domain

Nir2, like its Drosophila homolog retinal degeneration B (RdgB), contains an N-terminal phosphatidylinositol-transfer protein (PI-TP)-like domain. Previous studies have suggested that RdgB plays an important role in the fly phototransduction cascade and that its PI-transfer domain is critical for this function. In this domain, a specific mutation, T59E, induces a dominant retinal degeneration phenotype. Here we show that a similar mutation, T59E in the human Nir2 protein, targets Nir2 to spherical cytosolic structures identified as lipid droplets by the lipophilic dye Nile red. A truncated Nir2T59E mutant consisting of only the PI-transfer domain was also targeted to lipid droplets, whereas neither the wild-type Nir2 nor the Nir2T59A mutant was associated with lipid droplets under regular growth conditions. However, oleic-acid treatment caused translocation of wild-type Nir2, but not translocation of the T59A mutant, to lipid droplets. This treatment also induced partial targeting of endogenous Nir2, which is mainly associated with the Golgi apparatus, to lipid droplets. Targeting of Nir2 to lipid droplets was attributed to its enhanced threonine phosphorylation. These results suggest that a specific threonine within the PI-transfer domain of Nir2 provides a regulatory site for targeting to lipid droplets. In conjunction with the role of PI-TPs in lipid transport, this targeting may affect intracellular lipid trafficking and distribution and may provide the molecular basis underlying the dominant effect of the RdgB-T59E mutant on retinal degeneration.

Specific phosphorylation and activation of ERK1c by MEK1b: a unique route in the ERK cascade

Extracellular signal-regulated kinases (ERKs) are key signaling molecules that regulate a large number of cellular processes, including mitosis. We showed previously that ERK1c, an alternatively spliced form of ERK1, facilitates mitotic Golgi fragmentation without the involvement of ERK1 and ERK2. Here we demonstrate that activation of ERK1c is mainly mediated by mitogen-activated protein kinase (MAPK)/ERK kinase 1b (MEK1b), which is an alternatively spliced form of MEK1 that was previously considered an inactive kinase. MEK1b phosphorylation and activity are preferentially stimulated by nocodazole, to induce its specific activity toward ERK1c. MEK1/2, on the other hand, preferentially target ERK1/2 in response to growth factors, such as EGF. As previously demonstrated for ERK1c, also MEK1b expression and activity are elevated during mitosis, and thereby enhance Golgi fragmentation and mitotic rate. MEK1 activity is also increased during mitosis, but this isoform facilitates mitotic progression without affecting the Golgi architecture. These results illustrate that the ERK cascade is divided into two routes: the classic MEK1/2-ERK1/2 and the splice-variant MEK1b-ERK1c, each of which regulates distinct cellular processes and thus extends the cascade specificity.

Hierarchic and cooperative binding of the rat liver nuclear protein C/EBP at the hepatitis B virus enhancer.

We used the enhancer-binding protein C/EBP as a model to study the nature and the complexity of interaction of an enhancer-binding protein with its target DNA. We found that bacterially expressed C/EBP binds the hepatitis B virus enhancer at multiple sites in a hierarchic and cooperative manner. At low concentrations, only the E element is occupied, but at higher concentrations, additional sites are filled including a site that binds EP, a crucial enhancer-activating protein. This pattern of C/EBP binding may explain the concentration-dependent effect of C/EBP on enhancer activity.

The Detection of MAPK Signaling

Mitogen‐activated protein kinase (MAPK) cascades are central pathways that participate in the intracellular transmission of extracellular signals. Each of the MAPK signaling cascades seems to consist of three to five tiers of protein kinases that sequentially activate each other by phosphorylation. Since the majority of MAPK cascade components are kinases, the methods used to detect their activation involve determining phosphorylation state and protein kinase activities. The Basic Protocol describes the use of immunoblotting with specific anti‐phospho antibody to detect activation of MAPK components. Alternative methods described are immunoprecipitation of desired protein kinases followed by phosphorylation of specific substrates and the use of an in‐gel kinase assay. These methods have proven useful in the study of the MAPK signaling cascades.