Dynamic phosphorylation of tyrosine 665 in pseudopodium-enriched atypical kinase 1 (PEAK1) is essential for the regulation of cell migration and focal adhesion turnover.

Pseudopodium-enriched atypical kinase 1 (PEAK1) is a recently described tyrosine kinase that associates with the actin cytoskeleton and focal adhesion (FA) in migrating cells. PEAK1 is known to promote cell migration, but the responsible mechanisms remain unclear. Here, we show that PEAK1 controls FA assembly and disassembly in a dynamic pathway controlled by PEAK1 phosphorylation at Tyr-665. Knockdown of endogenous PEAK1 inhibits random cell migration. In PEAK1-deficient cells, FA lifetimes are decreased, FA assembly times are shortened, and FA disassembly times are extended. Phosphorylation of Tyr-665 in PEAK1 is essential for normal PEAK1 localization and its function in the regulation of FAs; however, constitutive phosphorylation of PEAK1 Tyr-665 is also disruptive of its function, indicating a requirement for precise spatiotemporal regulation of PEAK1. Src family kinases are required for normal PEAK1 localization and function. Finally, we provide evidence that PEAK1 promotes cancer cell invasion through Matrigel by a mechanism that requires dynamic regulation of Tyr-665 phosphorylation.

Pseudopodium-enriched atypical kinase 1 regulates the cytoskeleton and cancer progression [corrected].

Regulation of the actin-myosin cytoskeleton plays a central role in cell migration and cancer progression. Here, we report the discovery of a cytoskeleton-associated kinase, pseudopodium-enriched atypical kinase 1 (PEAK1). PEAK1 is a 190-kDa nonreceptor tyrosine kinase that localizes to actin filaments and focal adhesions. PEAK1 undergoes Src-induced tyrosine phosphorylation, regulates the p130Cas-Crk-paxillin and Erk signaling pathways, and operates downstream of integrin and epidermal growth factor receptors (EGFR) to control cell spreading, migration, and proliferation. Perturbation of PEAK1 levels in cancer cells alters anchorage-independent growth and tumor progression in mice. Notably, primary and metastatic samples from colon cancer patients display amplified PEAK1 levels in 81% of the cases. Our findings indicate that PEAK1 is an important cytoskeletal regulatory kinase and possible target for anticancer therapy.

The Rho-family GEF Asef2 activates Rac to modulate adhesion and actin dynamics and thereby regulate cell migration.

Asef2 is a recently identified Rho-family guanine nucleotide exchange factor (GEF) that has been implicated in the modulation of actin, but its function in cell migration and adhesion dynamics is not well understood. In this study, we show that Asef2 is an important regulator of cell migration and adhesion assembly and disassembly (turnover). Asef2 localizes with actin at the leading edge of cells. Knockdown of endogenous Asef2 impairs migration and significantly slows the turnover of adhesions. Asef2 enhances both Rac1 and Cdc42 activity in HT1080 cells, but only Rac1 is crucial for the Asef2-promoted increase in migration and adhesion turnover. Phosphoinositide 3-kinase (PI3K) and the serine/threonine kinase Akt are also essential for the Asef2-mediated effects on migration and adhesion turnover. Consistent with this, Asef2 increases the amount of active Akt at the leading edge of cells. Asef2 signaling leads to an overall decrease in Rho activity, which is crucial for stimulating migration and adhesion dynamics. Thus, our results reveal an important new role for Asef2 in promoting cell migration and rapid adhesion turnover by coordinately regulating the activities of Rho-family GTPases.

Myeloid cell receptor LRP1/CD91 regulates monocyte recruitment and angiogenesis in tumors.

Recruitment of monocytes into sites of inflammation is essential in the immune response. In cancer, recruited monocytes promote invasion, metastasis, and possibly angiogenesis. LDL receptor-related protein (LRP1) is an endocytic and cell-signaling receptor that regulates cell migration. In this study, we isografted PanO2 pancreatic carcinoma cells into mice in which LRP1 was deleted in myeloid lineage cells. Recruitment of monocytes into orthotopic and subcutaneous tumors was significantly increased in these mice, compared with control mice. LRP1-deficient bone marrow-derived macrophages (BMDM) expressed higher levels of multiple chemokines, including, most prominently, macrophage inflammatory protein-1α/CCL3, which is known to amplify inflammation. Increased levels of CCL3 were detected in LRP1-deficient tumor-associated macrophages (TAM), isolated from PanO2 tumors, and in RAW 264.7 macrophage-like cells in which LRP1 was silenced. LRP1-deficient BMDMs migrated more rapidly than LRP1-expressing cells in vitro. The difference in migration was reversed by CCL3-neutralizing antibody, by CCR5-neutralizing antibody, and by inhibiting NF-κB with JSH-23. Inhibiting NF-κB reversed the increase in CCL3 expression associated with LRP1 gene silencing in RAW 264.7 cells. Tumors formed in mice with LRP1-deficient myeloid cells showed increased angiogenesis. Although VEGF mRNA expression was not increased in LRP1-deficient TAMs, at the single-cell level, the increase in TAM density in tumors with LRP1-deficient myeloid cells may have allowed these TAMs to contribute an increased amount of VEGF to the tumor microenvironment. Our results show that macrophage density in tumors is correlated with cancer angiogenesis in a novel model system. Myeloid cell LRP1 may be an important regulator of cancer progression.