The Cytoskeletal Protein Cyclase-Associated Protein 1 (CAP1) in Breast Cancer: Context-Dependent Roles in Both the Invasiveness and Proliferation of Cancer Cells and Underlying Cell Signals.

As a conserved actin-regulating protein, CAP (adenylyl Cyclase-Associated Protein) functions to facilitate the rearrangement of the actin cytoskeleton. The ubiquitously expressed isoform CAP1 drives mammalian cell migration, and accordingly, most studies on the involvement of CAP1 in human cancers have largely been based on the rationale that up-regulated CAP1 will stimulate cancer cell migration and invasiveness. While findings from some studies reported so far support this case, lines of evidence largely from our recent studies point to a more complex and profound role for CAP1 in the invasiveness of cancer cells, where the potential activation of cell adhesion signaling is believed to play a key role. Moreover, CAP1 was also found to control proliferation in breast cancer cells, through the regulation of ERK (External signal-Regulated Kinase). Alterations in the activities of FAK (Focal Adhesion Kinase) and ERK from CAP1 depletion that are consistent to the opposite adhesion and proliferation phenotypes were detected in the metastatic and non-metastatic breast cancer cells. In this review, we begin with the overview of the literature on CAP, by highlighting the molecular functions of mammalian CAP1 in regulating the actin cytoskeleton and cell adhesion. We will next discuss the role of the FAK/ERK axis, and possibly Rap1, in mediating CAP1 signals to control breast cancer cell adhesion, invasiveness, and proliferation, largely based on our latest findings. Finally, we will discuss the relevance of these novel mechanistic insights to ultimately realizing the translational potential of CAP1 in targeted therapeutics for breast cancer.

Phosphorylation of the cytoskeletal protein CAP1 controls its association with cofilin and actin.

Cell signaling can control the dynamic balance between filamentous and monomeric actin by modulating actin regulatory proteins. One family of actin regulating proteins that controls actin dynamics comprises cyclase-associated proteins 1 and 2 (CAP1 and 2, respectively). However, cell signals that regulate CAPs remained unknown. We mapped phosphorylation sites on mouse CAP1 and found S307 and S309 to be regulatory sites. We further identified glycogen synthase kinase 3 as a kinase phosphorylating S309. The phosphomimetic mutant S307D/S309D lost binding to its partner cofilin and, when expressed in cells, caused accumulation of actin stress fibers similar to that in cells with reduced CAP expression. In contrast, the non-phosphorylatable S307A/S309A mutant showed drastically increased cofilin binding and reduced binding to actin. These results suggest that the phosphorylation serves to facilitate release of cofilin for a subsequent cycle of actin filament severing. Moreover, our results suggest that S307 and S309 function in tandem; neither the alterations in binding cofilin and/or actin, nor the defects in rescuing the phenotype of the enlarged cell size in CAP1 knockdown cells was observed in point mutants of either S307 or S309. In summary, we identify a novel regulatory mechanism of CAP1 through phosphorylation.

Identification and characterization of Csh3 as an SH3 protein that interacts with fission yeast Cap1.

Schizosaccharomyces pombe Cap1 has been identified as the (adenylyl) cyclase-associated protein. Cap1 was able to bind Cap1 itself and actin. Cap1 localized at the growing tip, and this localization was dependent on the Cap1 P2 region. In a two-hybrid screening using cap1 as bait, we isolated csh3, which encodes a protein of 296 amino acids with an SH3 domain and a proline/glutamine-rich region. The binding of Csh3 and Cap1 was confirmed by in vivo pull down assays. Cooperative functions of Csh3 and Cap1 were observed. Deletion of both csh3 and cap1 resulted in heightened sensitivity to CaCl2, while disruption of either gene alone did not have any effect in this regard. In addition, over-expression of csh3 or cap1 alone did not affect cell growth, while over-expression of both genes resulted in growth retardation. Finally, while Csh3-GFP localized to the cytoplasm in wild-type cells, its localization was altered in cap1Δ cells, suggesting that the interaction between Csh3 and Cap1 controls the cellular localization of Csh3. These results demonstrate that Cap1 in Schizo. pombe is a multifunctional protein that functions through interaction with Cap1 itself and other proteins including adenylyl cyclase, actin and Csh3.

Mammalian adenylyl cyclase-associated protein 1 (CAP1) regulates cofilin function, the actin cytoskeleton, and cell adhesion.

CAP (adenylyl cyclase-associated protein) was first identified in yeast as a protein that regulates both the actin cytoskeleton and the Ras/cAMP pathway. Although the role in Ras signaling does not extend beyond yeast, evidence supports that CAP regulates the actin cytoskeleton in all eukaryotes including mammals. In vitro actin polymerization assays show that both mammalian and yeast CAP homologues facilitate cofilin-driven actin filament turnover. We generated HeLa cells with stable CAP1 knockdown using RNA interference. Depletion of CAP1 led to larger cell size and remarkably developed lamellipodia as well as accumulation of filamentous actin (F-actin). Moreover, we found that CAP1 depletion also led to changes in cofilin phosphorylation and localization as well as activation of focal adhesion kinase (FAK) and enhanced cell spreading. CAP1 forms complexes with the adhesion molecules FAK and Talin, which likely underlie the cell adhesion phenotypes through inside-out activation of integrin signaling. CAP1-depleted HeLa cells also had substantially elevated cell motility as well as invasion through Matrigel. In summary, in addition to generating in vitro and in vivo evidence further establishing the role of mammalian CAP1 in actin dynamics, we identified a novel cellular function for CAP1 in regulating cell adhesion.

Application of three-dimensional printing in cardiovascular diseases: a bibliometric analysis.

AIM: This paper aimed to explore the application of three-dimensional (3D) printing in cardiovascular diseases, to reach an insight in this field and prospect the future trend. METHODS: The articles were selected from the Web of Science Core Collection database. Excel 2019, VOSviewer 1.6.16, and CiteSpace 6.1.R6 were used to analyze the information. RESULTS: A total of 467 papers of 3D printing in cardiovascular diseases were identified, and the first included literature appeared in 2000. A total of 692 institutions from 52 countries participated in the relevant research, while the United States of America contributed to 160 articles and were in a leading position. The most productive institution was Curtin University , and Zhonghua Sun who has posted the most articles ( n =8) was also from there. The Frontiers in Cardiovascular Medicine published most papers ( n =25). The Journal of Thoracic and Cardiovascular Surgery coveted the most citations ( n =520). Related topics of frontiers will still focus on congenital heart disease, valvular heart disease, and left atrial appendage closure. CONCLUSIONS: The authors summarized the publication information of the application of 3D printing in cardiovascular diseases related literature from 2000 to 2023, including country and institution of origin, authors, and publication journal. This study can reflect the current hotspots and novel directions for the application of 3D printing in cardiovascular diseases.

CAP1 (cyclase-associated protein 1) mediates the cyclic AMP signals that activate Rap1 in stimulating matrix adhesion of colon cancer cells.

We previously reported that CAP1 (Cyclase-Associated Protein 1) regulates matrix adhesion in mammalian cells through FAK (Focal Adhesion Kinase). More recently, we discovered a phosphor-regulation mechanism for CAP1 through the Ser307/Ser309 tandem site that is of critical importance for all CAP1 functions. However, molecular mechanisms underlying the CAP1 function in adhesion and its regulation remain largely unknown. Here we report that Rap1 also facilitates the CAP1 function in adhesion, and more importantly, we identify a novel signaling pathway where CAP1 mediates the cAMP signals, through the cAMP effectors Epac (Exchange proteins directly activated by cAMP) and PKA (Protein Kinase A), to activate Rap1 in stimulating matrix adhesion in colon cancer cells. Knockdown of CAP1 led to opposite adhesion phenotypes in SW480 and HCT116 colon cancer cells, with reduced matrix adhesion and reduced FAK and Rap1 activities in SW480 cells while it stimulated matrix adhesion as well as FAK and Rap1 activities in HCT116 cells. Importantly, depletion of CAP1 abolished the stimulatory effects of the cAMP activators forskolin and isoproterenol, as well as that of Epac and PKA, on matrix adhesion in both cell types. Our results consistently support a required role for CAP1 in the cAMP activation of Rap1. Identification of the key role for CAP1 in linking the major second messenger cAMP to activation of Rap1 in stimulating adhesion, which may potentially also regulate proliferation in other cell types, not only vertically extends our knowledge on CAP biology, but also carries important translational potential for targeting CAP1 in cancer therapeutics.

Gold Nanorod Substrate for Rat Fetal Neural Stem Cell Differentiation into Oligodendrocytes.

Gold nanorods (AuNRs) have been proposed to promote stem cell differentiation in vitro and in vivo. In this study, we examined a particular type of AuNR in supporting the differentiation of rat fetal neural stem cells (NSCs) into oligodendrocytes (ODCs). AuNRs were synthesized according to the seed-mediated method resulting in nanorods with an aspect ratio of around 3 (~12 nm diameter, 36 nm length) and plasmon resonance at 520 and 780 nm, as confirmed by transmission electron microscopy (TEM) and UV-vis spectroscopy, respectively. A layer-by-layer approach was used to fabricate the AuNR substrate on the functionalized glass coverslips. NSCs were propagated for 10 days using fibroblast growth factor, platelet-derived growth-factor-supplemented culture media, and differentiated on an AuNR or poly-D-lysine (PDL)-coated surface using differentiation media containing triiodothyronine for three weeks. Results showed that NSCs survived better and differentiated faster on the AuNRs compared to the PDL surface. By week 1, almost all cells had differentiated on the AuNR substrate, whereas only ~60% differentiated on the PDL surface, with similar percentages of ODCs and astrocytes. This study indicates that functionalized AuNR substrate does promote NSC differentiation and could be a viable tool for tissue engineering to support the differentiation of stem cells.

Dynamic Phosphorylation and Dephosphorylation of Cyclase-Associated Protein 1 by Antagonistic Signaling through Cyclin-Dependent Kinase 5 and cAMP Are Critical for the Protein Functions in Actin Filament Disassembly and Cell Adhesion.

Cyclase-associated protein 1 (CAP1) is a conserved actin-regulating protein that enhances actin filament dynamics and also regulates adhesion in mammalian cells. We previously found that phosphorylation at the Ser307/Ser309 tandem site controls its association with cofilin and actin and is important for CAP1 to regulate the actin cytoskeleton. Here, we report that transient Ser307/Ser309 phosphorylation is required for CAP1 function in both actin filament disassembly and cell adhesion. Both the phosphomimetic and the nonphosphorylatable CAP1 mutant, which resist transition between phosphorylated and dephosphorylated forms, had defects in rescuing the reduced rate of actin filament disassembly in the CAP1 knockdown HeLa cells. The phosphorylation mutants also had defects in alleviating the elevated focal adhesion kinase (FAK) activity and the enhanced focal adhesions in the knockdown cells. In dissecting further phosphoregulatory cell signals for CAP1, we found that cyclin-dependent kinase 5 (CDK5) phosphorylates both Ser307 and Ser309 residues, whereas cAMP signaling induces dephosphorylation at the tandem site, through its effectors protein kinase A (PKA) and exchange proteins directly activated by cAMP (Epac). No evidence supports an involvement of activated protein phosphatase in executing the dephosphorylation downstream from cAMP, whereas preventing CAP1 from accessing its kinase CDK5 appears to underlie CAP1 dephosphorylation induced by cAMP. Therefore, this study provides direct cellular evidence that transient phosphorylation is required for CAP1 functions in both actin filament turnover and adhesion, and the novel mechanistic insights substantially extend our knowledge of the cell signals that function in concert to regulate CAP1 by facilitating its transient phosphorylation.

c-Abl phosphorylates Dok1 to promote filopodia during cell spreading.

Filopodia are dynamic F-actin structures that cells use to explore their environment. c-Abl tyrosine kinase promotes filopodia during cell spreading through an unknown mechanism that does not require Cdc42 activity. Using an unbiased approach, we identified Dok1 as a specific c-Abl substrate in spreading fibroblasts. When activated by cell adhesion, c-Abl phosphorylates Y361 of Dok1, promoting its association with the Src homology 2 domain (SH2)/SH3 adaptor protein Nck. Each signaling component was critical for filopodia formation during cell spreading, as evidenced by the finding that mouse fibroblasts lacking c-Abl, Dok1, or Nck had fewer filopodia than cells reexpressing the product of the disrupted gene. Dok1 and c-Abl stimulated filopodia in a mutually interdependent manner, indicating that they function in the same signaling pathway. Dok1 and c-Abl were both detected in filopodia of spreading cells, and therefore may act locally to modulate actin. Our data suggest a novel pathway by which c-Abl transduces signals to the actin cytoskeleton through phosphorylating Dok1 Y361 and recruiting Nck.

Phosphorylation Regulates CAP1 (Cyclase-Associated Protein 1) Functions in the Motility and Invasion of Pancreatic Cancer Cells.

Pancreatic cancer has the worst prognosis among major malignancies, largely due to its highly invasive property and difficulty in early detection. Mechanistic insights into cancerous transformation and especially metastatic progression are imperative for developing novel treatment strategies. The actin-regulating protein CAP1 is implicated in human cancers, while the role still remains elusive. In this study, we investigated roles for CAP1 and its phosphor-regulation in pancreatic cancer cells. No evidence supports remarkable up-regulation of CAP1 in the panel of cancer cell lines examined. However, knockdown of CAP1 in cancer cells led to enhanced stress fibers, reduced cell motility and invasion into Matrigel. Phosphorylation of CAP1 at the S308/S310 tandem regulatory site was elevated in cancer cells, consistent with hyper-activated GSK3 reported in pancreatic cancer. Inhibition of GSK3, a kinase for S310, reduced cell motility and invasion. Moreover, phosphor mutants had defects in alleviating actin stress fibers and rescuing the reduced invasiveness in the CAP1-knockdown PANC-1 cells. These results suggest a required role for transient phosphorylation for CAP1 function in controlling cancer cell invasiveness. Depletion of CAP1 also reduced FAK activity and cell adhesion, but did not cause significant alterations in ERK or cell proliferation. CAP1 likely regulates cancer cell invasiveness through effects on both actin filament turnover and cell adhesion. Finally, the growth factor PDGF induced CAP1 dephosphorylation, suggesting CAP1 may mediate extracellular signals to control cancer cell invasiveness. These findings may ultimately help develop strategies targeting CAP1 or its regulatory signals for controlling the invasive cycle of the disease.

Phosphorylation of the Cytoskeletal Protein CAP1 Regulates Non-Small Cell Lung Cancer Survival and Proliferation by GSK3ß.

Adenylate cyclase-associated protein 1 (CAP1) is an evolutionarily conserved protein that regulates actin dynamics. Our previous study indicates that CAP1 is overexpressed in NSCLC tissues and correlated with poor clinical outcomes. Further establishing the role and dissecting underlying mechanisms are imperative before targeting CAP1 can become a possibility for cancer treatment. Here we report our findings that knockdown of CAP1 inhibited cell proliferation and induced apoptosis in vitro and in vivo. Moreover, phosphor mutants of CAP1 at the S307/S309 regulatory site had compromised rescue effects for both the invasiveness and the proliferation in CAP1-knockdown cells and GSK3ß kinase inhibitor LiCl inhibited cell phosphorylation site S307/S309 by up-regulating the expression of p53, BAK, BAD and cleaved PARP induced ROS production, decreased lung cancer cell viability, adhesion, proliferation, migration and invasion, and induction of apoptosis. These novel mechanistic insights may ultimately open up avenues for strategies targeting CAP1 in the treatment of lung cancer, tailored for specific types of the highly diverse disease.

CDK5 Functions as a Tumor Promoter in Human Lung Cancer.

Cyclin-dependent kinase 5 (CDK5), an atypical member of the cyclin-dependent kinase family, plays an important role in the nervous system. Recent studies have shown that CDK5 is also associated with tumors. However, few studies have been done to investigate the mechanism underlying the connection between CDK5 and cancers. To explore the role of CDK5 in cancers by using an extensive bioinformatics data mining process. We mined the transcriptional, survival, functions and structure of CDK5 gene through databases and in vitro experiments. We found that higher CDK5 expression levels in most cancer cell lines while lower expression in liver and brain cancer cell lines. High expression of CDK5 was associated with shorter overall survival (OS) in lung cancer. In addition, high expression level of CDK5 promoted lung cancer cells proliferation and metastasis. Inhibited CDK5 decreases CAP1 phosphorylation. CDK5 may prove to be a valid target of anticancer therapies.

Akt phosphorylation of serine 21 on Pak1 modulates Nck binding and cell migration.

The p21-activated protein kinases (Paks) regulate cellular proliferation, differentiation, transformation, and survival through multiple downstream signals. Paks are activated directly by the small GTPases Rac and Cdc42 and several protein kinases including Akt and PDK-1. We found that Akt phosphorylated and modestly activated Pak1 in vitro. The major site phosphorylated by Akt on Pak1 mapped to serine 21, a site originally shown to be weakly autophosphorylated on Pak1 when Cdc42 or Rac activates it. A peptide derived from the region surrounding serine 21 was a substrate for Akt but not Pak1 in vitro, and Akt stimulated serine 21 phosphorylation on the full-length Pak1 much better than Rac did. The adaptor protein Nck binds Pak near serine 21, and its association is regulated by phosphorylation of this site. We found that either treatment of Pak1 in vitro with Akt or coexpression of constitutively active Akt with Pak1 reduced Nck binding to Pak1. In HeLa cells, green fluorescent protein-tagged Pak1 was concentrated at focal adhesions and was released when Akt was cotransfected. A peptide containing the Nck binding site of Pak1 fused to a portion of human immunodeficiency virus Tat to allow it to enter cells was used to test the functional importance of Nck/Pak binding in Akt-stimulated cell migration. This Tat-Nck peptide reduced Akt-stimulated cell migration. Together, these data suggest that Akt modulates the association of Pak with Nck to regulate cell migration.

CAP1 (Cyclase-Associated Protein 1) Exerts Distinct Functions in the Proliferation and Metastatic Potential of Breast Cancer Cells Mediated by ERK.

The actin-regulating protein CAP1 is implicated in the invasiveness of human cancers. However, the exact role remains elusive and controversial given lines of conflicting evidence. Moreover, a potential role in the proliferative transformation has largely been overlooked. Further establishing the role and dissecting underlying mechanisms are imperative before targeting CAP1 can become a possibility for cancer treatment. Here we report our findings that CAP1 exerts cell type-dependent functions in the invasiveness of breast cancer cells. Depletion of CAP1 in the metastatic MDA-MB-231 and BT-549 cancer cells stimulated the metastatic potential while it actually inhibited it in the non-metastatic MCF-7 cancer cells or in normal cells. Moreover, we demonstrate functions for CAP1 in cancer cell proliferation and anchorage-independent growth, again in a cell context-dependent manner. Importantly, we identify pivotal roles for the ERK-centered signaling in mediating both CAP1 functions. Phosphor mutants of CAP1 at the S307/S309 regulatory site had compromised rescue effects for both the invasiveness and proliferation in CAP1-knockdown cells, suggesting that CAP1 likely mediates upstream cell signals to control both functions. These novel mechanistic insights may ultimately open up avenues for strategies targeting CAP1 in the treatment of breast cancer, tailored for specific types of the highly diverse disease.

Mammalian CAP (Cyclase-associated protein) in the world of cell migration: Roles in actin filament dynamics and beyond.

Cell migration is essential for a variety of fundamental biological processes such as embryonic development, wound healing, and immune response. Aberrant cell migration also underlies pathological conditions such as cancer metastasis, in which morphological transformation promotes spreading of cancer to new sites. Cell migration is driven by actin dynamics, which is the repeated cycling of monomeric actin (G-actin) into and out of filamentous actin (F-actin). CAP (Cyclase-associated protein, also called Srv2) is a conserved actin-regulatory protein, which is implicated in cell motility and the invasiveness of human cancers. It cooperates with another actin regulatory protein, cofilin, to accelerate actin dynamics. Hence, knockdown of CAP1 slows down actin filament turnover, which in most cells leads to reduced cell motility. However, depletion of CAP1 in HeLa cells, while causing reduction in dynamics, actually led to increased cell motility. The increases in motility are likely through activation of cell adhesion signals through an inside-out signaling. The potential to activate adhesion signaling competes with the negative effect of CAP1 depletion on actin dynamics, which would reduce cell migration. In this commentary, we provide a brief overview of the roles of mammalian CAP1 in cell migration, and highlight a likely mechanism underlying the activation of cell adhesion signaling and elevated motility caused by depletion of CAP1.

Analysis of expressed sequence tags (ESTs) from Lentinula edodes.

The 1,031 expressed sequence tags (ESTs) from the basidiomycete Lentinula edodes were generated as a pilot experiment to see distribution of genes expressed in L. edodes. Among them, genes for hydrophobin, which are specifically found in filamentous fungi, were the most frequently obtained ESTs (33 times), suggesting that they are highly expressed in L. edodes. In addition to known hydrophobin 1 and 2 types, our analysis revealed the existence of novel types of hydrophobin, which we named hydrophobin 3, 4, and 5. The second and the third most highly obtained ESTs were phosphatidylserine decarboxylase and formate dehydrogenase, which were obtained eight and seven times, respectively. It should be noted that two important genes (argonaute and RNA-dependent RNA polymerase) involved in the RNAi pathway were found, suggesting a future application for gene knock-down by RNA interference. The 53 ESTs were identical with the sequences already reported in L. edodes. The 433 ESTs were found to show significant sequence similarity (E value <1 x 10(-5)) with the proteins reported (or predicted) in other species. In total, 387,952 bp were sequenced and registered in DDBJ/GenBank (accession number BJ998097-BJ999127).

Mitochondrial shuttling of CAP1 promotes actin- and cofilin-dependent apoptosis.

Mitochondria play a central role in regulating apoptosis by releasing proapoptotic contents such as cytochrome c, and generating reactive oxygen species (ROS). Early in apoptosis, proteins translocate to mitochondria to promote the release of their contents. Here, we show that the actin- and cofilin-interacting protein CAP1 has a role in apoptosis. When we induced apoptosis, CAP1 rapidly translocated to the mitochondria independently of caspase activation. Translocation was proapoptotic because CAP1-knockdown cells were resistant to apoptosis inducers. Overexpression of wild-type CAP1 did not stimulate apoptosis on its own, but stimulated cofilin-induced apoptosis. Apoptosis induction required a mitochondrial-targeting domain, localized in the N-terminus and also the actin-binding domain in the C-terminus. Taken together, these studies suggest that CAP1 provides a direct link from the actin cytoskeleton to the mitochondria by functioning as an actin shuttle.

Opposing roles for Akt1 and Akt2 in Rac/Pak signaling and cell migration.

The Akt/PKB isoforms have different roles in animals, with Akt2 primarily regulating metabolic signaling and Akt1 regulating growth and survival. Here we show distinct roles for Akt1 and Akt2 in mouse embryo fibroblast cell migration and regulation of the cytoskeleton. Akt1-deficient cells responded poorly to platelet-derived growth factor while Akt2-deficient cells had a dramatically enhanced response, resulting in a substantial increase in dorsal ruffling. Swapping domains between Akt1 and Akt2 demonstrated that the N-terminal region containing the pleckstrin homology domain and a linker region distinguishes the two isoforms, while the catalytic domains are interchangeable. Akt2 knock-out cells also migrated faster than wild-type cells, especially through extracellular matrix (ECM), while Akt1 knock-out cells migrated more slowly than wild-type cells. Consistently, Akt2 knock-out cells had elevated Pak1 and Rac activities, suggesting that Akt2 inhibits Rac and Pak1. Both Akt2 and Akt1 associated in complexes with Pak1, but only Akt2 inhibited Pak1 in kinase assays, suggesting an underlying molecular basis for the different cellular phenotypes. Together these data provide evidence for an unexpected functional link between Akt2 and Pak1 that opposes the actions of Akt1 on cell migration.

cGMP-dependent protein kinase phosphorylates p21-activated kinase (Pak) 1, inhibiting Pak/Nck binding and stimulating Pak/vasodilator-stimulated phosphoprotein association.

Endothelial cells are normally non-motile and quiescent; however, endothelial cells will become permeable and invade and proliferate to form new blood vessels (angiogenesis) in response to wounding, cancer, diabetic retinopathy, age-related macular degeneration, or rheumatoid arthritis. p21-activated kinase (Pak), an effector for the Rho GTPases Rac and Cdc42, is required for angiogenesis and regulates endothelial cell permeability and motility. Although Pak is primarily activated by Rac and Cdc42, there are additional proteins that regulate Pak activity and localization, including three AGC protein kinase family members, Akt-1, PDK-1, and cAMP-dependent protein kinase. We describe phosphorylation and regulation of Pak localization by a fourth AGC kinase family member, cGMP-dependent protein kinase (PKG). Using in vitro mapping, a phosphospecific antibody, co-transfection assays, and untransfected bovine aortic endothelial cells we determined that PKG phosphorylates Pak at serine 21. Phosphorylation was accompanied by changes in proteins associated with Pak. The adaptor protein Nck was released, whereas a novel complex with vasodilator-stimulated phosphoprotein was stimulated. Furthermore Ser-21 phosphorylation of Pak appears to be important for regulation of cell morphology. In both human umbilical vein endothelial cells and HeLa cells, activation of PKG in the presence of Pak stimulated tail retraction and cell polarization. However, in cells expressing S21A mutant Pak1, PKG activation or treatment with a peptide that blocks Nck/Pak binding caused aberrant cell morphology, blocked cell retraction, and mislocalized Pak, producing uropod (tail-like) structures. These data suggest that PKG regulates Pak and that the interaction plays a role in tail retraction.