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. 2016 Apr 21;127(16):1967-75.
doi: 10.1182/blood-2016-01-693572. Epub 2016 Mar 1.

p21-activated Kinase 2 Regulates HSPC Cytoskeleton, Migration, and Homing via CDC42 Activation and Interaction With β-Pix

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Free PMC article

p21-activated Kinase 2 Regulates HSPC Cytoskeleton, Migration, and Homing via CDC42 Activation and Interaction With β-Pix

Pavankumar N G Reddy et al. Blood. .
Free PMC article

Abstract

Cytoskeletal remodeling of hematopoietic stem and progenitor cells (HSPCs) is essential for homing to the bone marrow (BM). The Ras-related C3 botulinum toxin substrate (Rac)/cell division control protein 42 homolog (CDC42) effector p21-activated kinase (Pak2) has been implicated in HSPC homing and engraftment. However, the molecular pathways mediating Pak2 functions in HSPCs are unknown. Here, we demonstrate that both Pak2 kinase activity and its interaction with the PAK-interacting exchange factor-β (β-Pix) are required to reconstitute defective ITALIC! Pak2 (ITALIC! Δ/Δ)HSPC homing to the BM. Pak2 serine/threonine kinase activity is required for stromal-derived factor-1 (SDF1α) chemokine-induced HSPC directional migration, whereas Pak2 interaction with β-Pix is required to regulate the velocity of HSPC migration and precise F-actin assembly. Lack of SDF1α-induced filopodia and associated abnormal cell protrusions seen in ITALIC! Pak2 (ITALIC! Δ/Δ)HSPCs were rescued by wild-type (WT) Pak2 but not by a Pak2-kinase dead mutant (KD). Expression of a β-Pix interaction-defective mutant of Pak2 rescued filopodia formation but led to abnormal F-actin bundles. Although CDC42 has previously been considered an upstream regulator of Pak2, we found a paradoxical decrease in baseline activation of CDC42 in ITALIC! Pak2 (ITALIC! Δ/Δ)HSPCs, which was rescued by expression of Pak2-WT but not by Pak2-KD; defective homing of ITALIC! Pak2-deleted HSPCs was rescued by constitutive active CDC42. These data demonstrate that both Pak2 kinase activity and its interaction with β-Pix are essential for HSPC filopodia formation, cytoskeletal integrity, and homing via activation of CDC42. Taken together, we provide mechanistic insights into the role of Pak2 in HSPC migration and homing.

Figures

Figure 1
Figure 1
Validation and retroviral expression of Pak2 biochemical mutants in HSPCs. (A) Schematic of polycistronic MSCV retroviral vectors expressing Cre and WT Pak2 or mutants with EGFP. (B) PCR analysis of genomic DNA extracted from FACS-sorted EGFP+ LSK cells harvested from Pak2fl/fl mice that were transduced with the indicated vectors; untransduced cells were used as a control. The positions of the WT and deleted bands are shown on the left and size ladder is shown on right. (C) Immunoblot to detect Pak2 expression in FACS-sorted EGFP+ LSK from WT (control) or Pak2fl/fl mice transduced with Cre alone (Pak2-deleted, Pak2Δ/Δ) or Cre-T2A-Pak2-WT (Pak2-WT). β-actin is used as a loading control. (D) In vitro kinase assay using MBP as a substrate and HA-tag immunoprecipitates from 32D cells transduced with vectors expressing Pak2-WT or Pak2 mutants. The blot showing phosphorylated threonine (p-Thr) and serine (p-Ser) residues and MBP indicates presence of substrate in all conditions. Bottom panel shows exogenous (HA) and total Pak2 expression from cell lysates with β-actin as a loading control. (E) Coimmunoprecipitates of HA-tagged Pak2-WT, Pak2-KD, and Pak2-Δβ-Pix from transduced 32D cell lysates analyzed by immunoblot for β-Pix. β-Pix expression in total cell lysates and HA blot of immunoprecipitates were used as loading controls. Ψ, Ψ packaging signal; EGFP, enhanced green fluorescent protein; IB, immunoblot; IP, immunoprecipitation; LTR, long terminal repeat; TCL, total cell lysate.
Figure 2
Figure 2
SDF1α-induced HSPC directional migration and velocity are regulated by the kinase activity of Pak2 and its interaction with β-Pix. WT and Pak2fl/fl LSK cells were transduced with indicated retroviruses, and FACS-sorted EGFP+ cells were imaged using a time-lapse microscope at 30-second intervals for 1 hour after the addition of SDF1α. The cells were tracked as described in “Methods” and analyzed using Metamorph software to generate tracks of migration for each individual cell and for quantification. (A) HSPC migration in response to SDF1α. Origins of all tracks were centered at the 0,0 XY coordinate with distance in micrometers on x- and y-axes shown (n > 11 per group). (B) Schematic representation of migration analysis; (C) the Euclidean distance in micrometers; (D) the velocity of migration in micrometers per second; and (E) the accumulated distance in micrometers. All data expressed as mean ± standard error of the mean (SEM). In panel E, only Pak2-Δβ-Pix is significantly different from control. One of the 3 independent experiments shown and the statistical significance was determined using the Mann-Whitney U test. *P < .05. n.s., not significant.
Figure 3
Figure 3
Pak2 regulates SDF1α- and integrin-induced filopodia formation in HSPCs in a kinase- and β-Pix–dependent manner. WT and Pak2fl/fl LSK cells were transduced with indicated retroviruses and FACS sorted. EGFP+ cells were stimulated with SDF1α and stained for F-actin using rhodamine-labeled phalloidin and counter-stained with DAPI. The slides were then imaged using Zeiss LSM 700 Laser Scanning confocal microscope with a 100× objective lens. (A) Representative images of Control (WT-Cre) and Pak2Δ/Δ. (B) Representative images of Pak2fl/fl cells transduced with Cre coexpressing Pak2-WT, Pak2-KD, and Pak2-Δβ-Pix mutants showing F-actin (red), DAPI (blue), overlap (merge). The boxed region is enlarged in grayscale in the inset to better define filopodia and abnormal protrusions containing F-actin. Red arrows indicate filopodia, white arrows indicate excessive protrusions, and yellow bars indicate abnormal F-actin bundles. The data are from 1 of 2 or more independent experiments each scoring at least 70 cells.
Figure 4
Figure 4
Pak2 regulates CDC42 activation. (A) WT and Pak2fl/fl LSK cells were transduced with indicated retroviruses and active GTP-bound CDC42 was determined by PBD pull-down assay in the FACS-sorted EGFP+ cells in control (WT) and Pak2fl/f cells and cells coexpressing Cre and Pak2-WT, Pak2-KD, and Pak2-Δβ-Pix. CDC42 from total cell lysates was used as a loading control. (B) Normalized densitometric values comparing GTP-bound to total CDC42 from panel B. (C) Representative images of sorted SDF1-stimulated EGFP+ LSK cells from WT (control) and Pak2 fl/fl (Pak2Δ/Δ) transduced as in panel B, showing F-actin (red), DAPI (blue), overlap (merge). The boxed region is enlarged in grayscale in the inset to show filopodia/abnormal protrusions. Red arrows indicate filopodia after stimulation with SDF1α, the white arrows indicate excessive protrusions, and the yellow arrows indicate spontaneous filopodia-like formations in unstimulated conditions. Data are from 1 of 2 independent experiments each with at least 50 cells.
Figure 5
Figure 5
Pak2 kinase activity and its interaction with β-Pix regulate HSPC homing and CA-CDC42 rescued defective homing of Pak2 deletion. (A) Homing of LSK BM cells. WT and Pak2fl/fl LSK cells were transduced with indicated retroviruses and 2 × 105 sorted EGFP+ cells were injected into lethally irradiated mice. Sixteen hours postinfusion, homing to the BM was measured after harvesting femur, tibia, and iliac crest bones. The homing efficiency of indicated genotypes is expressed as a mean of the percentage of control (WT LSK transduced with Cre) for each mouse with mean shown as horizontal bar, n = 12-27 mice per group. (B) Sorted EGFP+ LSK cells (2 × 105) from WT (control) and Pak2fl/fl mice were transduced with vectors expressing Cre alone (Pak2Δ/Δ) or Cre and CA-CDC42 and injected into lethally irradiated mice. Sixteen hours postinfusion, the homing to the BM was measured from cells harvested from femur, tibia, and iliac crest. The homing efficiency of individual animals of the indicated genotypes is shown as a percent of control with mean (horizontal bar) ± SEM of 5 to 13 mice per group. The statistical significance was determined using the Mann-Whitney U test. ****P < .0001, ***P < .001, **P < .01, *P < .05.
Figure 6
Figure 6
The engraftment defect of Pak2-deleted HSCs is rescued with full-length Pak2 cDNA. (A) Engraftment of LSK BM cells. LSK cells from WT (Control) and Pak2fl/fl mice were transduced with either Cre (Pak2Δ/Δ) or Cre-T2A-Pak2-WT (Pak2-WT) retrovirus. Sorted EGFP+ cells (1 × 105) were injected into lethally irradiated mice. Sixteen weeks posttransplantation, the percentage of EGFP-expressing cells in the peripheral blood was measured and the data plotted for each mouse with mean shown as horizontal bar, n = 7 to 11 mice per group. (B) Sorted EGFP+ LSK cells (1 × 105) from WT (control) and Pak2fl/fl (Pak2Δ/Δ) were transduced with vectors as in Figure 5B and injected into lethally irradiated mice. Sixteen weeks posttransplantation, the percentage of engrafted EGFP-expressing cells in peripheral blood was determined. The data show mean ± SEM, n = 3 to 6 mice per group. The statistical significance was determined using the Mann-Whitney U test. ***P < .001, **P < .01.
Figure 7
Figure 7
Model depicting Pak2 mechanism in HSPC function. Pak2 kinase activity mediates CDC42 activation to regulate filopodia formation, directional migration and its interaction with β-Pix regulates proper actin remodeling, velocity of migration to control homing, and engraftment of HSPCs in mice.

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