Novel p21-activated kinase-dependent protrusions characteristically formed at the edge of transformed cells

Heckman, Carol A.; Urban, Jason M.; Cayer, Marilyn L.; Li, Y; Boudreau, Nancy; Barnes, Julie; Plummer, Howard K.; Hall, Christine; Kozma, Robert; Lim, Louis

doi:10.1016/j.yexcr.2003.12.020

Cited by 15 publications

(40 citation statements)

References 101 publications

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“…Some of the cell protrusion structures are indeed bulkier than the filopodia normally seen in fibroblasts. These features, along with the corresponding spatial distribution of various actin-remodeling proteins, are reminiscent of the more recently reported morphological features indicative of neoplastic cells containing, for instance, protrusive structures that are characteristic of PAK1 overactivity (15). The cell edge feature, which contains the Cdc42-bound activated PAK1 in breast carcinoma cells, is bulkier than filopodia and possibly represents a recently reported form of PAK1-dependent protrusions in neoplastic cells (15).…”

Section: Discussionsupporting

confidence: 54%

Spatially Distinct Binding of Cdc42 to PAK1 and N-WASP in Breast Carcinoma Cells

Parsons

Monypenny²,

Ameer-Beg

et al. 2005

Molecular and Cellular Biology

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While a significant amount is known about the biochemical signaling pathways of the Rho family GTPase Cdc42, a better understanding of how these signaling networks are coordinated in cells is required. In particular, the predominant subcellular sites where GTP-bound Cdc42 binds to its effectors, such as p21-activated kinase 1 (PAK1) and N-WASP, a homolog of the Wiskott-Aldritch syndrome protein, are still undetermined. Recent fluorescence resonance energy transfer (FRET) imaging experiments using activity biosensors show inconsistencies between the site of local activity of PAK1 or N-WASP and the formation of specific membrane protrusion structures in the cell periphery. The data presented here demonstrate the localization of interactions by using multiphoton time-domain fluorescence lifetime imaging microscopy (FLIM). Our data here establish that activated Cdc42 interacts with PAK1 in a nucleotide-dependent manner in the cell periphery, leading to Thr-423 phosphorylation of PAK1, particularly along the lengths of cell protrusion structures. In contrast, the majority of GFP-N-WASP undergoing FRET with Cy3-Cdc42 is localized within a transferrin receptor-and Rab11-positive endosomal compartment in breast carcinoma cells. These data reveal for the first time distinct spatial association patterns between Cdc42 and its key effector proteins controlling cytoskeletal remodeling.Cytoskeletal remodelling is a highly dynamic, tightly regulated process which drives the formation of cell protrusions necessary for cell movement. Members of the Rho family of GTPases regulate cytoskeletal dynamics by cycling between inactive GDP-bound and active GTP-bound states. Cdc42 activation results in a localized increase in actin polymerization and the formation of filopodia. Immediately downstream of Cdc42 are p21-activated kinase 1 (PAK1) as well as the Wiskott-Aldrich syndrome protein (WASP) family of proteins, both of which are activated through the binding of GTP Cdc42 via the Cdc42-and Rac-binding domain (CRIB or PBD). PAK1 has been reported to localize to regions of cytoskeletal assembly, and the activated form can induce cytoskeletal remodeling at the leading edge via downstream events such as microtubule growth and stathmin phosphorylation (11, 60). A close correlation has been found between the PAK1 activity state and the baseline invasiveness of human breast cancer cells as well as breast tumor grades (52). Paradoxically, the same work demonstrated that breast carcinoma cells expressing the GTPase binding-deficient H83L, H86L PAK1 mutant exhibited extensive membrane ruffling in the cell periphery. The lamellipodia formation and membrane ruffling observed in the presence of PAK1 were subsequently demonstrated by others to be independent of PAK catalytic activity (13). The relationship between the local level of PAK1 activity and the formation of specific subcellular structures is therefore unclear.The WASP-family proteins and the Arp2/3 complex are important nucleators of new actin filaments in response to signals causing cel...

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Section: Discussionsupporting

confidence: 54%

Spatially Distinct Binding of Cdc42 to PAK1 and N-WASP in Breast Carcinoma Cells

Parsons

Monypenny²,

Ameer-Beg

et al. 2005

Molecular and Cellular Biology

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“…As shown in previous studies (27,28,56), the phenotype of cells could be broken down by analysis of latent factors. Factors show a one-to-one correspondence to cell features.…”

Section: Cancer-type Reversal By Inhibitor Combinationsmentioning

confidence: 99%

Pattern analysis of microtubule-polymerizing and -depolymerizing agent combinations as cancer chemotherapies

Uppal

Li²,

Wendt³

et al. 2007

Int J Oncol

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Abstract. Subcellular distribution of mass can be analyzed by a technique that involves culturing cells on interferometers and digitizing their interference contours. Contour sampling resulted in 102 variables per cell, which were predictors of oncogenic transformation. Cell phenotypes can be deconstructed by use of latent factors, which represent the covariance of the real variables. The reversal of the cancertype phenotype by a combination of microtubule-stabilizing and -depolymerizing agents was described previously. The implications of these results have been explored by clinicians who treated patients with the combination of docetaxel and vinorelbine (Navelbine ® ). The current study was performed to determine the effects of different combinations on phenotype and in phases of the cell cycle other than mitosis. Combinations of paclitaxel with either colchicine, podophyllotoxin, nocodazole, or vinblastine caused phenotype reversal. Paclitaxel analogue, 7-deoxytaxol, by itself caused reversal. Factors #4, (filopodia), #5 (displacement and/or deep invaginations in the periphery), #8, and #12 took on values typical of normal cells, whereas the values of #7 (p21-activated kinase), and #13 (rounding up) shifted toward the cancer-type. All combinations altered microtubule arrangement at the cell edge. Delivery schedules and drug ratios used in clinical studies were subjected to analysis. Clinical response rates were better when the combination was not interspersed with a single agent (P=0.004). The results support the idea that efficacy depends upon simultaneous exposure to both agents, and suggest a novel mechanism for combination therapies. These therapies appear to restore in transformed cells some of the features of a contactinhibited cell, and to impede progress through the cell cycle even when provided at nanomolar concentrations.

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“…Although the organelle has not been reported in non-neuronal cells, we speculated that the FCs of filopodia would still differ qualitatively from those in other protrusions and, in particular, the large protrusions (LPs). Formation of the latter involves coordination over a broad expanse of cytoplasm, and previous work suggested that a mechanism may exist for coordinating FCs at the cell edge with FCs interior to the edge (Heckman et al, 2004). To test the postulated difference between FCs of filopodia and LPs, we used proteins or protein domains affecting PAK and GTPasemediated signaling since these were already implicated in LP formation.…”

mentioning

confidence: 99%

“…Since Nck binds to N-WASP and PAK via the same SH3[2] group (Lu et al, 1997;Rohatgi et al, 2001), it was possible that N-WASP might be titrating Nck and making it unavailable for binding PAK. If PAK were recruited to FCs by Nck, this could prevent LP formation (Heckman et al, 2004).…”

mentioning

confidence: 99%

“…PAK also affects aspects of cytoskeletal organization. Inhibition of PAK by the provision of kinase inhibitor domain (KID) caused stress fibers to reassemble on the FCs, but it was not known whether this was directly due to stabilization of the FC or to activation of actin contractile mechanisms Heckman et al, 2004). In the current research, we test the possibility that effectors of actin-FC integration, including PAK and Nck, affect formation of either filopodia or LPs.…”

mentioning

confidence: 99%

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Relationship of p21‐activated kinase (PAK) and filopodia to persistence and oncogenic transformation

Heckman

Demuth

Deters

et al. 2009

Journal Cellular Physiology

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Previously, we found that oncogenically transformed cells had fewer filopodia and more large, p21-activated kinase (PAK)-dependent features than normal cells. These large protrusions (LPs) were increased in cells expressing RhoA(N19) with Cdc42-associated kinase (ACK). Here, we determine how GTPase-mediated mechanisms of focal contact (FC) regulation affect these protrusions. Constructs encoding various proteins were introduced into cells which were then studied by microscopy and computerized image processing and analysis. Constructs that prevented PAK recruitment by PAK-interacting exchange factor (PIX) or restricted PAK residence time on FCs decreased both protrusions. Thus, filopodia were also PAK-dependent. A comparison of FC distribution in cells expressing PAK in the presence or absence of PAK kinase inhibitor domain (KID) suggested that PAK enlarged FCs without affecting the prevalence of either protrusion. KID or Nck expression increased LPs but not filopodia. Nck failed to synergize with KID or ACK and RhoA(N19) in enhancing LPs. Nck and KID synergistically enhanced filopodia, possibly because Nck recruited PAK to FCs while KID prevented their dissociation by PAK-mediated autophosphorylation. Coexpression of Nck, ACK, and RhoA(N19) abrogated filopodia and replicated the transformed phenotype. Since Nck recruitment of PAK is implicated in persistence of directional movement, we studied the PAK-Nck interface. Filopodia were eliminated by the Nck PAK-binding domain and LPs by the PAK Nck-binding domain. The results suggested that filopodia formation has more stringent requirements than LP formation, and Nck and PAK are used differently in the protrusions. Loss of filopodia in transformed cells may reflect defective regulation of GTPase mechanisms.

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Novel p21-activated kinase-dependent protrusions characteristically formed at the edge of transformed cells

Cited by 15 publications

References 101 publications

Spatially Distinct Binding of Cdc42 to PAK1 and N-WASP in Breast Carcinoma Cells

Spatially Distinct Binding of Cdc42 to PAK1 and N-WASP in Breast Carcinoma Cells

Pattern analysis of microtubule-polymerizing and -depolymerizing agent combinations as cancer chemotherapies

Relationship of p21‐activated kinase (PAK) and filopodia to persistence and oncogenic transformation

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