Polarity of the microtubule (MT) cytoskeleton is essential for many cell functions. Cytoplasmic linker–associated proteins (CLASPs) are MT-associated proteins thought to organize intracellular MTs and display a unique spatiotemporal regulation. In migrating epithelial cells, CLASPs track MT plus ends in the cell body but bind along MTs in the lamella. In this study, we demonstrate that glycogen synthase kinase 3β (GSK3β) directly phosphorylates CLASPs at multiple sites in the domain required for MT plus end tracking. Although complete phosphorylation disrupts both plus end tracking and association along lamella MTs, we show that partial phosphorylation of the identified GSK3β motifs determines whether CLASPs track plus ends or associate along MTs. In addition, we find that expression of constitutively active GSK3β destabilizes lamella MTs by disrupting lateral MT interactions with the cell cortex. GSK3β-induced lamella MT destabilization was partially rescued by expression of CLASP2 with mutated phosphorylation sites. This indicates that CLASP-mediated stabilization of peripheral MTs, which likely occurs in the vicinity of focal adhesions, may be regulated by local GSK3β inactivation.

Stearoyl-acyl carrier protein Delta(9) desaturase (Delta9D) produces oleic acid, a nutritionally valuable fatty acid containing a cis double bond between C-9 and C-10. This multiprotein diiron enzyme complex reacts with stearoyl-acyl carrier protein, reduced [2Fe-2S] ferredoxin, and O(2) to complete the highly regiospecific and stereoselective desaturation reaction. Interactions with the acyl chain provide stability to the enzyme-substrate complex, give an energetic contribution to catalytic selectivity, and help to order the electron transfer, O(2) binding, and C-H bond cleavage steps of catalysis. Reactions with natural acyl chains indicate the involvement of a highly reactive diiron intermediate capable of oxidizing secondary C-H bonds (bond dissociation energy approximately 95 kcal/mol), but also capable of diagnostic O-atom transfer reactions with the appropriate substrate analogues. For soluble Delta9D, the natural reaction may initiate at the C-10 position, in contrast to the well-established initial reactivity of the membrane enzyme homologue stearoyl-coenzyme A (CoA) Delta(9) desaturase at the C-9 position.

c p21-activated kinases (Paks) have been shown to regulate cytoskeleton rearrangements, cell proliferation, attachment, and migration in a variety of cellular contexts, including endothelial cells. However, the role of endothelial Pak in embryo development has not been reported, and currently, there is no consensus on the endothelial function of individual Pak isoforms, in particular p21-activated kinase 2 (Pak2), the main Pak isoform expressed in endothelial cells. In this work, we employ genetic and molecular studies that show that Pak2, but not Pak1, is a critical mediator of development and maintenance of endothelial cell function. Endothelial depletion of Pak2 leads to early embryo lethality due to flawed blood vessel formation in the embryo body and yolk sac. In adult endothelial cells, Pak2 depletion leads to severe apoptosis and acute angiogenesis defects, and in adult mice, endothelial Pak2 deletion leads to increased vascular permeability. Furthermore, ubiquitous Pak2 deletion is lethal in adult mice. We show that many of these defects are mediated through a newly unveiled Pak2/Bmk1 pathway. Our results demonstrate that endothelial Pak2 is essential during embryogenesis and also for adult blood vessel maintenance, and they also pinpoint the Bmk1/ Erk5 pathway as a critical mediator of endothelial Pak2 signaling.T he p21-activated kinases (Paks) are evolutionarily conserved serine/threonine kinases that act downstream of the Rho family GTPases, Rac1 and Cdc42, to regulate numerous cellular processes. p21-activated kinase 1 (Pak1) to Pak3, the group I Paks, are expressed in numerous tissues with Pak1 being predominantly expressed in brain, muscle, gastrointestinal tract, and thyroid and Pak3 being predominantly expressed in brain (1, 2). Compared to the more restricted expression of Pak1 and Pak3, Pak2 is ubiquitously expressed (3-9). Group I Pak family members share a high degree of homology (3), but they may play distinct roles as observed by the significantly different phenotypes of knockout (KO) animal models. While Pak1 KO and Pak3 KO mice are viable (10, 11), Pak2 KO mice are embryonic lethal at embryonic day 8.5 (E8.5) due to multiple developmental abnormalities, including cardiovascular defects (12).The role of Pak in endothelial cell (EC) signaling has been studied in animal models (5, 13) and cultured cells (6,10,14,15). Pak signaling is critical in regulating EC attachment, migration, and lumen formation (4-9). Furthermore, Paks have been implicated in maintaining the integrity of the endothelial barrier, but conflicting data implicate various Pak isoforms as both positive and negative regulators of maintaining barrier function (5, 13, 14, 16).A few in vivo studies have specifically implicated Pak2 in vascular pathways. A study in Danio rerio showed that pak2a, one of two Pak2 orthologs in this organism, plays an important role in cerebral vascular maintenance (5). In this study, chemical mutagenesis of the pak2a gene and pak2a-targeting morpholinos caused cerebral hemorrhage, implicating this...

Structure-based methods were used to design a potent and highly selective group II p21-activated kinase (PAK) inhibitor with a novel binding mode, compound 17. Hydrophobic interactions within a lipophilic pocket past the methionine gatekeeper of group II PAKs approached by these type I 1/2 binders were found to be important for improving potency. A structure-based hypothesis and strategy for achieving selectivity over group I PAKs, and the broad kinome, based on unique flexibility of this lipophilic pocket, is presented. A concentration-dependent decrease in tumor cell migration and invasion in two triple-negative breast cancer cell lines was observed with compound 17.

Epithelial cell migration is a complex process crucial for embryonic development, wound healing and tumor metastasis. It depends on alterations in cell-cell adhesion and integrin-extracellular matrix interactions and on actomyosin-driven, polarized leading edge protrusion. The small GTPase Rap is a known regulator of integrins and cadherins that has also been implicated in the regulation of actin and myosin, but a direct role in cell migration has not been investigated. Here, we report that activation of endogenous Rap by cAMP results in an inhibition of HGF-and TGFβ-induced epithelial cell migration in several model systems, irrespective of the presence of E-cadherin adhesion. We show that Rap activation slows the dynamics of focal adhesions and inhibits polarized membrane protrusion. Importantly, forced integrin activation by antibodies does not mimic these effects of Rap on cell motility, even though it does mimic Rap effects in short-term cell adhesion assays. From these results, we conclude that Rap inhibits epithelial cell migration, by modulating focal adhesion dynamics and leading edge activity. This extends beyond the effect of integrin affinity modulation and argues for an additional function of Rap in controlling the migration machinery of epithelial cells.