Pak4 Induces Premature Senescence via a Pathway Requiring p16(INK4)/p19(ARF) and Mitogen-Activated Protein Kinase Signaling

Exposure of primary cells to mitogenic stimuli or oncogenes often causes them to undergo premature senescence. This is most likely a protective function that prevents uncontrolled proliferation. Pak4 is a target for the Rho GTPase Cdc42. Pak4 is overexpressed in human tumor cell lines, and it is the only member of the Pak family that is highly transforming in immortalized fibroblasts. Here we show that in primary fibroblasts, activated Pak4 inhibits cell proliferation and promotes premature senescence. Furthermore, Pak4 expression levels are upregulated in response to stimuli that promote senescence. Pak4-induced arrest appears to be mediated by a pathway that requires the ERK mitogen-activated protein kinase, as well as the cell cycle inhibitors p16(INK4) and p19(ARF). These new results describing a role for Pak4 in senescence are important for understanding why this protein is associated with cancer and how it promotes transformation in immortalized cells

Mice Lacking the ERK1 Isoform of MAP Kinase Are Unimpaired in Emotional Learning

The extracellular signal-regulated kinases (ERKs) are members of the mitogen-activated protein kinase (MAPK) superfamily of enzymes and have recently garnered considerable attention in the field of learning and memory. ERK activation has been shown to be required for the induction of long-term potentiation (LTP) in the rat hippocampus and for the formation of associative and spatial memories in both the rat and the mouse. However, the individual roles for the two isoforms of ERK have yet to be deciphered. To investigate the specific contribution of the ERK1 (p44) isoform of MAPK to mammalian learning, we performed a general behavioral and physiological characterization of mice lacking the ERK1 gene. The ERK1-null animals demonstrated significantly higher levels of activity in the open field test. However, we observed no other discernible deficits in the ERK1 knockout mice in our behavioral testing. Specifically, no differences were observed in the acquisition or retention (24 h and 2 wk after training) of either contextual or cue fear conditioning between the ERK1(−/−) and their wild-type littermate controls. In addition, no learning phenotype was observed in the passive avoidance test. When hippocampal slices were analyzed, we found no deficits in baseline synaptic transmission or in tetanus-induced LTP in hippocampal area CA1. We found no apparent compensatory changes in the expression of ERK2 (p42 MAPK). We conclude that hippocampus- and amygdala-dependent emotional learning does not depend critically on the activity of ERK1

Functional deficits in PAK5, PAK6 and PAK5/PAK6 knockout mice.

The p21-activated kinases are effector proteins for Rho-family GTPases. PAK4, PAK5, and PAK6 are the group II PAKs associated with neurite outgrowth, filopodia formation, and cell survival. Pak4 knockout mice are embryonic lethal, while Pak5, Pak6, and Pak5/Pak6 double knockout mice are viable and fertile. Our previous work found that the double knockout mice exhibit locomotor changes and learning and memory deficits. We also found some differences with Pak5 and Pak6 single knockout mice and the present work further explores the potential differences of the Pak5 knockout and Pak6 knockout mice in comparison with wild type mice. The Pak6 knockout mice were found to weigh significantly more than the other genotypes. The double knockout mice were found to be less active than the other genotypes. The Pak5 knockout mice and the double knockout mice performed worse on the rotorod test. All the knockout genotypes were found to be less aggressive in the resident intruder paradigm. The double knockout mice were, once again, found to perform worse in the active avoidance assay. These results indicate, that although some behavioral differences are seen in the Pak5 and Pak6 single knockout mice, the double knockout mice exhibit the greatest changes in locomotion and learning and memory

A Role for ERK MAP Kinase in Physiologic Temporal Integration in Hippocampal Area CA1

Recent studies demonstrate a requirement for the Extracellular signal Regulated Kinase (ERK) mitogen-activated protein kinase (MAPK) cascade in both the induction of long-lasting forms of hippocampal synaptic plasticity and in hippocampus-dependent associative and spatial learning. In the present studies, we investigated mechanisms by which ERK might contribute to synaptic plasticity at Schaffer collateral synapses in hippocampal slices. We found that long-term potentiation (LTP) induced with a pair of 100-Hz tetani does not require ERK activation in mice whereas it does in rats. However, in mice, inhibition of ERK activation blocked LTP induced by two LTP induction paradigms that mimicked the endogenous θ rhythm. In an additional series of studies, we found that mice specifically deficient in the ERK1 isoform of MAPK showed no impairments in tests of hippocampal physiology. To investigate ERK-dependent mechanisms operating during LTP-inducing stimulation paradigms, we monitored spike production in the cell body layer of the hippocampus during the period of θ-like LTP-inducing stimulation. θ-burst stimulation (TBS) produced a significant amount of postsynaptic spiking, and the likelihood of spike production increased progressively over the course of the three trains of TBS independent of any apparent increase in Excitatory Post-Synaptic Potential (EPSP) magnitude. Inhibition of ERK activation dampened this TBS-associated increase in spiking. These data indicate that, for specific patterns of stimulation, ERK may function in the regulation of neuronal excitability in hippocampal area CA1. Overall, our data indicate that the progressive increase in spiking observed during TBS represents a form of physiologic temporal integration that is dependent on ERK MAPK activity

Summary of results and conclusions.

<p>WT = wild type mice, <i>Pak5</i>  =  <i>Pak5</i> knockout mice, <i>Pak6</i>  =  <i>Pak6</i> knockout mice. DKO =  <i>Pak5/Pak6</i> double knockout mice.</p

Figure 5

<p><b>a. Total Elevated Plus crosses</b>. The PAK5 knockout mice made more crosses into any arm of the elevated plus maze compared to the WT mice, the PAK6 knockout mice, and the DKO mice. The double knockout mice made significantly less crosses compared to the WT, PAK5 and PAK6 knockout mice. WT n = 8. PAK5 n = 5. PAK6 n = 8. DKO n = 5. <b>b. Open vs. Closed crosses.</b> The mice made significantly more crosses into the closed arms of the maze compared to the number of crosses they made into the open arms. (& = p<.05 compared to closed arms). WT n = 8. PAK5 n = 5. PAK6 n = 8. DKO n = 5. <b>c. </b><b>Elevated plus maze.</b> There was no difference in number of fecal boli between the genotypes. Only the PAK5 knockout mice jumped off the apparatus. WT n = 8. PAK5 n = 5. PAK6 n = 8. DKO n = 5.</p

Figure 1

<p><b>a. Total Body Weight Change</b>. PAK6 knockout mice had the largest increase in weight, significantly different from WT (a  =  p<.05 compared to WT), PAK5 (b =  p<.05 compared to PAK5), and DKO (d =  p<.05 compared to DKO). WT n = 8. PAK5 n = 5. PAK6 n = 8. DKO n = 5. <b>b. Body weight.</b> PAK6 knockout mice were significantly heavier than PAK5(b) and DKO(d) by 5 months of age and significantly heavier than WT(a) from 6 months of age. The DKO and PAK5 knockout mice weighed significantly less than the WT (a) and PAK6(c =  p<.05 compared to PAK6) at 5 months of age, but weighed similarly to the WT after 6 months of age. WT n = 8. PAK5 n = 5. PAK6 n = 8. DKO n = 5.</p

Figure 7

<p><b>a. Total touches to target and control cylinders</b>. The WT, PAK5 and PAK6 knockout mice made significantly more touches to the target cylinder compared to the control cylinder (* = p<.05 compared to control). The PAK6 knockout mice and the DKO mice made significantly less touches to the target cylinder compared to the number of touches the WT (a) and PAK5 knockout mice (b) made. The PAK6 knockout mice and the DKO mice made significantly less touches to the control cylinder compared to the WT mice. WT n = 8. PAK5 n = 5. PAK6 n = 8. DKO n = 5. <b>b. Percent touches to the target cylinder.</b> The double knockout mice had a significantly lower percentage of contact with the target cylinder compared to the WT, PAK5, and PAK6 knockout mice. WT n = 8. PAK5 n = 5. PAK6 n = 8. DKO n = 5.</p