Merlin Phosphorylation by p21-activated Kinase 2 and Effects of Phosphorylation on Merlin Localization

The Nf2 tumor suppressor gene product merlin is related to the membrane-cytoskeleton linker proteins of the band 4.1 superfamily, including ezrin, radixin, and moesin (ERMs). Merlin is regulated by phosphorylation in a Rac/cdc42-dependent fashion. We report that the phosphorylation of merlin at serine 518 is induced by the p21-activated kinase PAK2. This is demonstrated by biochemical fractionation, use of active and dominant-negative mutants of PAK2, and immunodepletion. By using wild-type and mutated forms of merlin and phospho-directed antibodies, we show that phosphorylation of merlin at serine 518 leads to dramatic protein relocalization. Neurofibromatosis type 2 (NF2)1 is an inherited disorder characterized by the development of Schwann cell tumors of the eighth cranial nerve. Mutations and loss of heterozygosity of theNF2 gene have been detected in NF2 patients and in various sporadic tumors, including schwannomas, meningiomas, and ependymomas (1). In further support of a role for NF2 in tumor suppression, mice heterozygous for an Nf2 mutation are predisposed to a wide variety of tumors with high metastatic potential (2). In a separate model in which Nf2 is inactivated specifically in Schwann cells, mice develop schwannomas and Schwann cell hyperplasia (3). The longest and predominant splice form of the Nf2gene codes for a 595-amino acid protein highly similar to the band 4.1 family of proteins. It is most closely related to the ERM proteins,moesin, ezrin, and radixin. The ERM proteins are thought to function as cell membrane-cytoskeleton linkers and are localized to cortical actin structures near the plasma membrane such as microvilli, membrane ruffles, and lamellipodia (4, 5). Likewise, merlin is localized to cortical actin structures, in patterns that partially overlap with the ERMs (1). It has been proposed that intramolecular binding of the N-terminal and C-terminal domains conformationally regulates the ERM proteins by masking binding sites for interacting proteins. The ERMs can also form homodimers and heterodimers, among themselves and with merlin, adding an additional level of complexity to the regulation of these proteins (6). The recently solved crystal structure of the moesin N/C-terminal complex strengthens this model of conformational regulation (7). Given the sequence and, most likely, structural similarities of merlin to the ERM proteins, it is possible that merlin itself could be regulated in a similar fashion. Recent studies (8, 9) have implicated additional factors in the regulation of the ERMs, including phospholipids and phosphorylation. Previous work from our group and others (10, 11) has shown that merlin is differentially phosphorylated as well and that merlin protein levels are affected by growth conditions such as cell confluency, loss of adhesion, or serum deprivation. Merlin is found in an hypophosphorylated form when the combination of cellular and environmental conditions are growth-inhibitory (10). ERMs can be phosphorylated by Rho kinase, and this phosphorylation can affect intramolecular association and cellular localization. Phosphorylation and/or phospholipids may promote the transition of the proteins to an active form by “opening” intra- and intermolecular associations. These active monomers can then bind to other interacting proteins and the actin cytoskeleton and induce actin-rich membrane projections (5,8, 12, 13). The induction of merlin phosphorylation by activated alleles of the Rho family GTPases has also been examined. Interestingly, although activated Rho did not induce noticeable phosphorylation of merlin, activated forms of Rac and cdc42 did. The site of Rac-induced phosphorylation was determined to be a serine at position 518; mutation of serine 518 results in reduced basal phosphorylation and eliminated Rac-induced phosphorylation (11). Although Rac and cdc42 are implicated in the regulation of many pathways, they are most associated with regulation of cytoskeleton reorganization and gene expression (for recent reviews see Refs.14-16). In light of the data demonstrating that activated Rac/cdc42 leads to phosphorylation and possible inactivation of merlin, the elucidation of the responsible effector pathways and their effects on merlin function are of major importance. Understanding this regulation of merlin could lead to a more complete appreciation of the effects of merlin loss in tumors

From Genetics to Biotechnology: Synthetic Biology as a Flexible Course-embedded Research Experience

The need for changing how science is taught and the expansion of undergraduate research experiences is essential to foster critical thinking in the Natural Sciences. Most faculty research programs only involve a small number of upper-level undergraduate students each semester. The course-based undergraduate research experience (CURE) model enables more students to take ownership over an independent project and experience authentic research. Further, by creating projects that fit into a curriculum\u27s learning goals and student-oriented outcomes, departments help strengthen critical thinking skills in the classroom. Here, we report on the incorporation of a synthetic biology CURE into a mid-level cellular biology course and two advanced level genetics/molecular biology courses. Synthetic biology involves systematic engineering of novel organisms, such as bacteria and plants, to work as functional devices to solve problems in medicine, agriculture, and manufacturing. The value of synthetic biology and its ultimate utility as a teaching tool relies on reusable, standard genetic parts that can be interchanged using common genetic engineering principles. This Synthetic biology CURE effectively achieves five essential goals: (1) a sense of project ownership; (2) self-efficacy: mastery of a manageable number of techniques; (3) increased tolerance for obstacles through challenging research; (4) increased communication skills; and (5) a sense of belonging in a larger scientific community. Based upon our student assessment data, we demonstrate that this course-based synthetic biology laboratory engages students directly in an authentic research experience and models important elements of collaboration, discovery, iteration, and critical thinking

Effects of fatiguing constant versus alternating intensity intermittent isometric muscle actions on maximal torque and neuromuscular responses

Objective: To determine the effects of constant versus alternating applications of torque during fatiguing, intermittent isometric muscle actions of the leg extensors on maximal voluntary isometric contraction (MVIC) torque and neuromuscular responses. Methods: Sixteen subjects performed two protocols, each consisting of 50 intermittent isometric muscle actions of the leg extensors with equal average load at a constant 60% MVIC or alternating 40 then 80% (40/80%) MVIC with a work-to-rest ratio of 6-s on and 2-s off. MVIC torque as well as electromyographic signals from the vastus lateralis (VL), vastus medialis (VM), and rectus femoris (RF) and mechanomyographic signals from the VL were recorded pretest, immediately posttest, and 5-min posttest. Results: The results indicated that there were no time-related differences between the 60% MVIC and 40/80% MVIC protocols. The MVIC torque decreased posttest (22 to 26%) and remained depressed 5-min posttest (9%). There were decreases in electromyographic frequency (14 to 19%) and mechanomyographic frequency (23 to 24%) posttest that returned to pretest levels 5-min posttest. There were no changes in electromyographic amplitude and mechanomyogrpahic amplitude. Conclusions: These findings suggested that these neuromuscular parameters did not track the fatigue-induced changes in MVIC torque after 5-min of recovery

Effects of fatiguing constant versus alternating intensity intermittent isometric muscle actions on maximal torque and neuromuscular responses

Objective: To determine the effects of constant versus alternating applications of torque during fatiguing, intermittent isometric muscle actions of the leg extensors on maximal voluntary isometric contraction (MVIC) torque and neuromuscular responses. Methods: Sixteen subjects performed two protocols, each consisting of 50 intermittent isometric muscle actions of the leg extensors with equal average load at a constant 60% MVIC or alternating 40 then 80% (40/80%) MVIC with a work-to-rest ratio of 6-s on and 2-s off. MVIC torque as well as electromyographic signals from the vastus lateralis (VL), vastus medialis (VM), and rectus femoris (RF) and mechanomyographic signals from the VL were recorded pretest, immediately posttest, and 5-min posttest. Results: The results indicated that there were no time-related differences between the 60% MVIC and 40/80% MVIC protocols. The MVIC torque decreased posttest (22 to 26%) and remained depressed 5-min posttest (9%). There were decreases in electromyographic frequency (14 to 19%) and mechanomyographic frequency (23 to 24%) posttest that returned to pretest levels 5-min posttest. There were no changes in electromyographic amplitude and mechanomyogrpahic amplitude. Conclusions: These findings suggested that these neuromuscular parameters did not track the fatigue-induced changes in MVIC torque after 5-min of recovery

Effect of sex on torque, recovery, EMG, and MMG responses to fatigue

The purpose of the present investigation was to examine the effect of sex on maximal voluntary isometric contraction (MVIC) torque and the EMG and MMG responses as a result of fatiguing, intermittent, submaximal (65% of MVIC), isometric elbow flexion muscle contractions. Methods: Eighteen men and women performed MVIC trials before (pretest), after (posttest), and 5-min after (5-min recovery) performing 50 intermittent, submaximal isometric muscle contractions. Surface electromyographic (EMG) and mechanomyographic (MMG) signals were simultaneously recorded from the biceps brachii muscle. Results: As a result of the fatiguing workbout torque decreased similarly from pretest to posttest for both the men (24.0%) and women (23.3%). After 5-min of recovery, torque had partially recovered for the men, while torque had returned to pretest levels for the women. For both sexes, from pretest to posttest EMG mean power frequency and MMG amplitude decreased, but returned to pretest levels after 5-min of recovery. Conclusions: In the present study, there were sex-related differences in muscle fatigue that were not associated with the EMG or MMG responses

Effect of sex on torque, recovery, EMG, and MMG responses to fatigue

The purpose of the present investigation was to examine the effect of sex on maximal voluntary isometric contraction (MVIC) torque and the EMG and MMG responses as a result of fatiguing, intermittent, submaximal (65% of MVIC), isometric elbow flexion muscle contractions. Methods: Eighteen men and women performed MVIC trials before (pretest), after (posttest), and 5-min after (5-min recovery) performing 50 intermittent, submaximal isometric muscle contractions. Surface electromyographic (EMG) and mechanomyographic (MMG) signals were simultaneously recorded from the biceps brachii muscle. Results: As a result of the fatiguing workbout torque decreased similarly from pretest to posttest for both the men (24.0%) and women (23.3%). After 5-min of recovery, torque had partially recovered for the men, while torque had returned to pretest levels for the women. For both sexes, from pretest to posttest EMG mean power frequency and MMG amplitude decreased, but returned to pretest levels after 5-min of recovery. Conclusions: In the present study, there were sex-related differences in muscle fatigue that were not associated with the EMG or MMG responses

Associations between residual feed intake and apparent nutrient digestibility, in vitro methane-producing activity, and volatile fatty acid concentrations in growing beef cattle

The objectives of this study were to examine the relationship between residual feed intake (RFI) and DM and nutrient digestibility, in vitro methane production, and volatile fatty acid (VFA) concentrations in growing beef cattle. Residual feed intake was measured in growing Santa Gertrudis steers (Study 1; n = 57; initial BW = 291.1 ± 33.8 kg) and Brangus heifers (Study 2; n = 468; initial BW = 271.4 ± 26.1 kg) fed a high-roughage-based diet (ME = 2.1 Mcal/kg DM) for 70 d in a Calan-gate feeding barn. Animals were ranked by RFI based on performance and feed intake measured from day 0 to 70 (Study 1) or day 56 (Study 2) of the trial, and 20 animals with the lowest and highest RFI were identified for subsequent collections of fecal and feed refusal samples for DM and nutrient digestibility analysis. In Study 2, rumen fluid and feces were collected for in vitro methane-producing activity (MPA) and VFA analysis in trials 2, 3, and 4. Residual feed intake classification did not affect BW or BW gain (P \u3e 0.05), but low-RFI steers and heifers both consumed 19% less (P \u3c 0.01) DMI compared with high-RFI animals. Steers with low RFI tended (P \u3c 0.1) to have higher DM digestibility (DMD) compared with high-RFI steers (70.3 vs. 66.5 ± 1.6% DM). Heifers with low RFI had 4% higher DMD (76.3 vs. 73.3 ± 1.0% DM) and 4 to 5% higher (P \u3c 0.01) CP, NDF, and ADF digestibility compared with heifers with high RFI. Low-RFI heifers emitted 14% less (P \u3c 0.01) methane (% GE intake; GEI) calculated according to Blaxter and Clapperton (1965) as modified by Wilkerson et al. (1995), and tended (P = 0.09) to have a higher rumen acetate:propionate ratio than heifers with high RFI (GEI = 5.58 vs. 6.51 ± 0.08%; A:P ratio = 5.02 vs. 4.82 ± 0.14%). Stepwise regression analysis revealed that apparent nutrient digestibilities (DMD and NDF digestibility) for Study 1 and Study 2 accounted for an additional 8 and 6%, respectively, of the variation in intake unaccounted for by ADG and mid-test BW0.75. When DMD, NDF digestibility, and total ruminal VFA were added to the base model for Study 2, trials 2, 3, and 4, the R2 increased from 0.33 to 0.47, explaining an additional 15% of the variation in DMI unrelated to growth and body size. On the basis of the results of these studies, differences in observed phenotypic RFI in growing beef animals may be a result of inter-animal variation in apparent nutrient digestibility and ruminal VFA concentrations