LIM Kinase 1 (LIMK1), a serine/threonine kinase, modulates actin polymerization and microtubule assembly. The function of LIMK1 is regulated by kinases that are activated by Rho and Rac GTPases. LIMK1 is overexpressed in various cancerous cell types and tissues and its overexpression promotes increased invasion and metastasis of breast and prostate cancer cells. Membrane-Type Matrix Metalloproteinase 1 (MT1-MMP) is a member of the zinc-binding collagenase family, which is involved in extracellular matrix breakdown and activation of secreted MMP-2. The balance between activation and inhibition of MT1- MMP and MMP-2 helps maintaining normal extracellular matrix turnover. However, it has been shown that elevated MT1-MMP expression can cause excessive ECM digestion and promote tumor invasion and metastasis. Since RhoA and Rac1 have been implicated in metastasis and invasion along with LIMK1 activation, we investigated a possible link between LIMK1 and MT1-MMP. Our results show that the level of MT1-MMP expression is correlated with that of LIMK1 and LIMK1 acts as a transcriptional regulator of MT1-MMP. Additionally, we show that LIMK1 physically associates with MT1-MMP and promotes its translocation to the plasma membrane.

The Hippo pathway is frequently deregulated in human cancers, but mutations and deletions of core signalome members are rare, suggesting that our understanding of its upstream regulators remains incomplete. A focused RNAi-based kinome screen identified novel candidate regulators of Hippo signaling, including STK25 and MST4. Here, we characterize the kinase STK25 as a novel upstream activator of LATS signaling. Depletion of STK25 was found to significantly reduce YAP phosphorylation in response to Hippo-activating stimuli, with consequent increases in YAP/TAZ activity and increased proliferation and resistance to cell cycle arrest. Mechanistically, STK25 activates LATS independently of the canonical MST/MAP4K axis, wherein STK25 directly promotes the phosphorylation of the LATS kinase activation loop in the absence of a preceding hydrophobic motif phosphorylation event. This differentiates STK25 from all other identified Hippo kinases to date, which may explain why singular loss of this kinase cannot be compensated for by the presence of other Hippo kinases. We also find that loss of STK25 increases YAP/TAZ signaling in vivo and that this promotes organ overgrowth in murine models. Interestingly, STK25 is frequently focally deleted in a spectrum of human cancers, suggesting that its loss might represent a way by which cancer cells functionally inactivate Hippo signaling. We also report that STK25 may be a novel regulator of mTOR signaling, as loss of STK25 hyper-activates mTOR signaling in response to amino acids and growth factors, but not to energy stresses. Interestingly, we find that MST4, a kinase closely related to STK25, appears to have highly context-specific Hippo regulatory functions; loss of MST4 was found to modulate Hippo signaling only in non-polarized cell lines, suggesting that polarity-responsive subcellular localization of MST4 may dictate its ability to interact with Hippo signaling. Lastly, we describe a novel role for Hippo signaling as a surveillance system for abnormal prolongation of mitosis, in which the LATS kinases regulate cell fate following abnormal mitosis via its control over p53-p21 signaling and YAP/TAZ signaling. This work thus identifies new roles and mechanisms by which kinases interact in the context of this major tumor suppressor pathway to control cellular processes critical to physiologic homeostasis. / 2021-10-07T00:00:00Z

This study examined effects of carbohydrate (C), carbohydrate-protein (CP), or placebo (P) beverages following eccentric resistance exercise on muscle damage by serum creatine kinase (CK), muscle protein breakdown by urinary 3-methylhistidine (3MH), muscle soreness, isokinetic muscle strength, muscle glycogen resynthesis, and serum hormones. Untrained males (N=26) underwent a 9-day controlled meat-free diet and 24 hr urine collections. To reduce glycogen, subjects cycled for 40 min at 70% of VO_2peakfollowed by 5 cycling sprints on day 4 evening. On day 5, fasted subjects performed 100 eccentric leg flexions at 120% of 1-RM and drank C (n=8, 1.25 g C/kg), CP (n=9, 0.875 g C/kg, 0.375 g protein/kg), or P (n=9) beverages immediate post-exercise (IPE) and 2 hr later. Muscle biopsies were taken IPE on day 5, and days 6 and 8 mornings. Blood was obtained days 4-10 fasted plus IPE, 3 hr, and 6 hr post-exercise on day 5. At 3 hr on day 5, insulin was higher for CP (24.6 ( 15.5 &amp;#181;IU/ml) and C (17.2 +/- 10.9 &amp;#181;IU/ml) than P (5.3 +/- 0.4 &amp;#181;IU/ml, p<.05). Glycogen was low on day 5, partially recovered on day 6, and normal by day 8 (p<.01) with no difference among groups. Isokinetic quadriceps peak torque at 60^o/s decreased 24% on day 6 and remained depressed by 21% on d 8 (p<.01) for all groups. Soreness peaked on day 7 and CK on day 8 (p<.01) with no differences by group. CK increased (p<.01) over day 5 (delta CP 24.6 +/- 19.1, delta P 39.2 +/- 71.6, delta C 70.8 +/- 60.4 U/L) and was highest for C (p<.05). On day 5, CP had lowest 3MH (193.0 +/- 13.8 &amp;#181;mol/d) versus C (251.1 +/- 22.5 &amp;#181;mol/d, p<.05). Testosterone at 3 hr on day 5 was lower for C (4.2 +/- 0.3 ng/ml) and CP (4.3 +/- 0.3 ng/ml) versus P (5.1 +/- 0.2 ng/ml, p<.05). In summary, glycogen, muscle strength and soreness were unaffected by beverage. However, a CP beverage may be beneficial for reducing muscle damage and protein breakdown on the day of eccentric resistance exercise. / Ph. D.

Aim: The purpose of this study was to investigate the influence of docosahexaenoic acid (DHA) on muscle damage and inflammation following an acute eccentric exercise bout. Methods: A double-blind placebo-controlled, study was performed using 41 healthy, untrained males aged 18-28 y who consumed either 2 g/d DHA or placebo (PL, corn oil) for 32 days. Supplements were consumed for 28 days prior to exercise. Participants completed an eccentric exercise procedure of the elbow flexors at 140% of 1-RM (6 sets x 10 repetitions). The time under tension (TUT) for each set of eccentric contractions was recorded manually from the investigators voice commands. Fasted blood samples for prostaglandin E2 (PGE2), interleukin-6 (IL-6), interleukin-1 receptor antagonist (IL1-ra), C-reactive protein and creatine kinase (CK) were assessed on days 1, 2 and 4. Fasted serum DHA was measured at baseline (day -28) and on day 1. Peak isometric strength of the elbow flexors, delayed-onset muscle soreness, and range of motion were measured on day 1 prior to exercise and days 2, 3, and 4 following exercise. Results: DHA significantly reduced natural log of CK (p<0.05) response over 4 d. Additionally, IL-6 area under the curve (AUC) was reduced for DHA compared to PL (3.6 ± 2.5 pg/mL vs. 5.3 ± 2.7 pg/mL) (p<0.05). TUT/set was higher in the DHA group compared to placebo (p<0.05). There were no other significant differences between treatments. Conclusion: DHA supplementation produced lower indicators of muscle damage (CK) and inflammation (IL-6 AUC). DHA supplementation resulted in greater TUT/set. / Master of Science

Atherosclerosis is a multifactorial inflammatory disease of the arterial wall and its clinical manifestations, including myocardial infarction and stroke, are the leading causes of death in western societies. Recent data has suggested that disruption of protein homeostasis in a cell's endoplasmic reticulum (ER), a condition known as ER stress, is associated with the progression of atherosclerosis. Furthermore, signaling by the serine/threonine kinase glycogen synthase kinase (GSK)-3α/β mediates pro-atherogenic processes. This thesis examines the role of ER stress and GSK3α/β signaling in atherosclerosis. Initially, three apolipoprotein-E deficient (ApoE-/-) mouse models of accelerated atherosclerosis were established. Relative to ApoE-/- mice fed a chow diet, pro-atherogenic conditions promoted hepatic steatosis, atherosclerosis, ER stress and GSK3β activity. A subset of mice from each group were given the GSK3α/β inhibitor valproate. Valproate supplementation suppressed hepatic steatosis, atherosclerosis and GSK3β activity in each mouse model without altering ER stress levels. This study revealed a role for ER stress and GSK3α/β in multiple murine models of atherosclerosis. Next, we investigated ER stress and GSK3α/β signaling in macrophage foam cell formation. In macrophages, ER stress induced GSK3α/β activity in a protein kinase R-like endoplasmic reticulum kinase (PERK) dependent manor. GSK3α/β inhibition attenuated ER stress induced lipid accumulation and the expression of distal components of the PERK pathway. Overexpression of constitutively active GSK3β induced foam cell formation. In mice, valproate supplementation attenuated PERK signaling in peritoneal macrophages and macrophages within atherosclerotic lesions. Together, these results point to GSK3α/β being a downstream component of the PERK pathway and that PERK-GSK3α/β signaling mediates ER stress induced foam cell formation. Lastly, we investigated the tissue and homolog specific functions of GSK3α and GSK3β in atherosclerosis. In high fat diet (HFD) fed low-density lipoprotein receptor deficient (LDLR-/-) mice, deletion of GSK3α or GSK3β in hepatocytes did not alter liver lipid content or atherosclerosis. Myeloid cell deletion of GSK3α, but not GSK3β, attenuated HFD induced atherosclerosis. Mechanistically, deletion of GSK3α in macrophages promotes the anti-atherogenic M2 macrophage phenotype by modulating signal transducer and activator of transcription (STAT)-3 and STAT6 phosphorylation and activation. Together, the data presented in this thesis suggest; 1) GSK3α/β inhibition attenuates atherosclerosis in multiple mouse models, 2) PERK-GSK3α/β signaling regulates macrophage foam cell formation and 3) myeloid cell GSK3α mediates atherosclerosis and macrophage phenotype. / Thesis / Doctor of Philosophy (PhD)

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