p21-Activated kinase 4 promotes prostate cancer progression through CREB.

Prostate cancer is initially androgen-dependent but, over time, usually develops hormone- and chemo-resistance. The present study investigated a role for p21-activated kinase 4 (PAK4) in prostate cancer progression. PAK4 activation was markedly inhibited by H89, a specific protein kinase A (PKA) inhibitor, and PAK4 was activated by the elevation of cAMP. The catalytic subunit of PKA interacted with the regulatory domain of PAK4, and directly phosphorylated PAK4 at serine 474 (S474). Catalytically active PAK4 enhanced the transcriptional activity of CREB independent of S133 phosphorylation. Stable knockdown of PAK4 in PC-3 and DU145 prostate cancer cells inhibited tumor formation in nude mice. Decreased tumorigenicity correlated with decreased expression of CREB and its targets, including Bcl-2 and cyclin A1. Additionally, in androgen-dependent LNCap-FGC cells, PAK4 regulated cAMP-induced neuroendocrine differentiation, which is known to promote tumor progression. Finally, PAK4 enhanced survival and decreased apoptosis following chemotherapy. These results suggested that PAK4 regulates progression toward hormone- and chemo-resistance in prostate cancer, and this study identified both a novel activation mechanism and potential downstream effector pathways. Therefore, PAK4 may be a promising therapeutic target in prostate cancer.

Identification of a calmodulin-regulated soybean Ca(2+)-ATPase (SCA1) that is located in the plasma membrane.

Ca(2)+-ATPases are key regulators of Ca(2+) ion efflux in all eukaryotes. Animal cells have two distinct families of Ca(2+) pumps, with calmodulin-stimulated pumps (type IIB pumps) found exclusively at the plasma membrane. In plants, no equivalent type IIB pump located at the plasma membrane has been identified at the molecular level, although related isoforms have been identified in non-plasma membrane locations. Here, we identify a plant cDNA, designated SCA1 (for soybean Ca(2+)-ATPase 1), that encodes Ca(2+)-ATPase and is located at the plasma membrane. The plasma membrane localization was determined by sucrose gradient and aqueous two-phase membrane fractionations and was confirmed by the localization of SCA1p tagged with a green fluorescent protein. The Ca(2+)-ATPase activity of the SCA1p was increased approximately sixfold by calmodulin (K(1/2) approximately 10 nM). Two calmodulin binding sequences were identified in the N-terminal domain. An N-terminal truncation mutant that deletes sequence through the two calmodulin binding sites was able to complement a yeast mutant (K616) that was deficient in two endogenous Ca(2+) pumps. Our results indicate that SCA1p is structurally distinct from the plasma membrane-localized Ca(2+) pump in animal cells, belonging instead to a novel family of plant type IIB pumps found in multiple subcellular locations. In plant cells from soybean, expression of this plasma membrane pump was highly and rapidly induced by salt (NaCl) stress and a fungal elicitor but not by osmotic stress.

Competitive binding of calmodulin isoforms to calmodulin-binding proteins: implication for the function of calmodulin isoforms in plants.

In plants, multiple calmodulin (CaM) isoforms exist in an organism which vary in their primary structures in as much as 32 residues out of their 148 amino acids. These CaM isoforms show differences in their expression patterns and/or target enzyme activation ability. To further understand the biological significance of CaM isoforms, we examined whether CaM isoforms act on specific regulatory targets. In gel overlay assays on various soybean tissue extracts, surprisingly, two soybean CaM isoforms (SCaM-1 and SCaM-4) did not show significant differences in their target binding protein profiles, although they exhibited minor differences in their relative target binding affinities. In addition, both SCaM isoforms not only effectively bound five known plant CaMBPs, but also showed competitive binding to these proteins. Finally, immunolocalization experiments with the SCaM proteins in sections of various tissues using specific antibodies revealed similar distribution patterns for the SCaM isoforms except for root tissues, which indicates that the SCaM isoforms are concomitantly expressed in most plant tissues. These results suggest that CaM isoforms may compete for binding to CaMBPs in vivo. This competitive nature of CaM isoforms may allow modulation of Ca(2+)/CaM signaling pathways by virtue of relative abundance and differential target activation potency.

Involvement of specific calmodulin isoforms in salicylic acid-independent activation of plant disease resistance responses.

The Ca2+ signal is essential for the activation of plant defense responses, but downstream components of the signaling pathway are still poorly defined. Here we demonstrate that specific calmodulin (CaM) isoforms are activated by infection or pathogen-derived elicitors and participate in Ca2+-mediated induction of plant disease resistance responses. Soybean CaM (SCaM)-4 and SCaM-5 genes, which encode for divergent CaM isoforms, were induced within 30 min by a fungal elicitor or pathogen, whereas other SCaM genes encoding highly conserved CaM isoforms did not show such response. This pathogen-triggered induction of these genes specifically depended on the increase of intracellular Ca2+ level. Constitutive expression of SCaM-4 and SCaM-5 in transgenic tobacco plants triggered spontaneous induction of lesions and induces an array of systemic acquired resistance (SAR)-associated genes. Surprisingly, these transgenic plants have normal levels of endogenous salicylic acid (SA). Furthermore, coexpression of nahG gene did not block the induction of SAR-associated genes in these transgenic plants, indicating that SA is not involved in the SAR gene induction mediated by SCaM-4 or SCaM-5. The transgenic plants exhibit enhanced resistance to a wide spectrum of virulent and avirulent pathogens, including bacteria, fungi, and virus. These results suggest that specific CaM isoforms are components of a SA-independent signal transduction chain leading to disease resistance.

Reciprocal regulation of mammalian nitric oxide synthase and calcineurin by plant calmodulin isoforms.

Calmodulin (CaM) is the primary mediator of Ca signal transduction processes in cells. Unlike animal cells, plant cells express multiple CaM isoforms. One cloned soybean CaM isoform (SCaM-4) half-maximally activated mammalian nitric oxide synthase (NOS) at 180 nM while another (SCaM-1) served as a competitive antagonist (Ki approximately 120 nM) of this activation. The reciprocal was true for the protein phosphatase calcineurin (CaN); SCaM-1 half-maximally activated mammalian CaN at approximately 12 nM, and SCaM-4 competitively antagonized (Ki approximately 70 nM) its activation. The reciprocal enzyme activation and competitive inhibition exhibited by these plant CaM isoforms suggest that their differential expression in cells could allow selective activation of some target enzymes and the selective inhibition of others. This may allow for a branching or bifurcation in the Ca2+-CaM signal transduction pathway and to alterations in cell function.

Differential activation of NAD kinase by plant calmodulin isoforms. The critical role of domain I.

NAD kinase is a Ca2+/calmodulin (CaM)-dependent enzyme capable of converting cellular NAD to NADP. The enzyme purified from pea seedlings can be activated by highly conserved soybean CaM, SCaM-1, but not by the divergent soybean CaM isoform, SCaM-4 (Lee, S. H., Kim, J. C., Lee, M. S., Heo, W. D., Seo, H. Y., Yoon, H. W., Hong, J. C., Lee, S. Y., Bahk, J. D., Hwang, I., and Cho, M. J. (1995) J. Biol. Chem. 270, 21806-21812). To determine which domains were responsible for this differential activation of NAD kinase, a series of chimeric SCaMs were generated by exchanging functional domains between SCaM-4 and SCaM-1. SCaM-4111, a chimeric SCaM-1 that contains the first domain of SCaM-4, was severely impaired (only 40% of maximal) in its ability to activate NAD kinase. SCaM-1444, a chimeric SCaM-4 that contains the first domain of SCaM-1 exhibited nearly full ( approximately 70%) activation of NAD kinase. Only chimeras containing domain I of SCaM-1 produced greater than half-maximal activation of NAD kinase. To define the amino acid residue(s) in domain I that were responsible for this differential activation, seven single residue substitution mutants of SCaM-1 were generated and tested for NAD kinase activation. Among these mutants, only K30E and G40D showed greatly reduced NAD kinase activation. Also a double residue substitution mutant, K30E/G40D, containing these two mutations in combination was severely impaired in its NAD kinase-activating potential, reaching only 20% of maximal activation. Furthermore, a triple mutation, K30E/M36I/G40D, completely abolished NAD kinase activation. Thus, our data suggest that domain I of CaM plays a key role in the differential activation of NAD kinase exhibited by SCaM-1 and SCaM-4. Further, the residues Lys30 and Glu40 of SCaM-1 are critical for this function.

Identification of a novel divergent calmodulin isoform from soybean which has differential ability to activate calmodulin-dependent enzymes.

Calmodulin plays pivotal roles in the transduction of various Ca(2+)-mediated signals and is one of the most highly conserved proteins in eukaryotic cells. In plants, multiple calmodulin isoforms with minor amino acid sequence differences were identified but their functional significances are unknown. To investigate the biological function of calmodulins in the regulation of calmodulin-dependent enzymes, we cloned cDNAs encoding calmodulins in soybean. Among the five cDNAs isolated from soybean, designated as SCaM-1 to -5, SCaM-4 and -5 encoded very divergent calmodulin isoforms which have 32 amino acid substitutions from the highly conserved calmodulin, SCaM-1 encoded by SCaM-1 and SCaM-3. SCaM-4 protein produced in Escherichia coli showed typical characteristics of calmodulin such as Ca(2+)-dependent electrophoretic mobility shift and the ability to activate phosphodiesterase. However, the extent of mobility shift and antigenicity of SCaM-4 were different from those of SCaM-1. Moreover, SCaM-4 did not activate NAD kinase at all in contrast to SCaM-1. Also there were differences in the expression pattern of SCaM-1 and SCaM-4. Expression levels of SCaM-4 were approximately 5-fold lower than those of SCaM-1 in apical and elongating regions of hypocotyls. In addition, SCaM-4 transcripts were barely detectable in root whereas SCaM-1 transcripts were as abundant as in apical and elongating regions of hypocotyls. In conclusion, the different biochemical properties together with differential expression of SCaM-4 suggest that this novel calmodulin may have different functions in plant cells.