Arthur M. Edelman

5′-AMP Activates the AMP-activated Protein Kinase Cascade, and Ca2+/Calmodulin Activates the Calmodulin-dependent Protein Kinase I Cascade, via Three Independent Mechanisms

AMP-activated protein kinase (AMPK) and Ca2+/calmodulin (CaM)-dependent protein kinase I (CaMKI) are protein kinases that are regulated both by allosteric activation (AMP and Ca2+/CaM, respectively) and by phosphorylation by upstream protein kinases (AMPK kinase (AMPKK) and CaMKI kinase (CaMKIK), respectively). We now report that AMPKK can activate CaMKI and that, conversely, CaMKIK can activate AMPK. CaMKIK is 68-fold more effective at activating CaMKI than AMPK, while AMPKK is 17-fold more effective at activating AMPK than CaMKI. Our results suggest that CaMKIK and AMPKK are distinct enzymes dedicated to their respective kinase targets but with some overlap in their substrate specificities. The availability of alternative substrates for AMPKK and CaMKIK allowed the unequivocal demonstration that AMP and Ca2+/calmodulin promote the activation of AMPK and CaMKI, respectively, via three independent mechanisms: 1) direct activation of AMPK and CaMKI, 2) activation of AMPKK and CaMKIK, and 3) by binding to AMPK and CaMKI, inducing exposure of their phosphorylation sites. Since AMP and Ca2+/calmodulin each has a triple effect in its respective system, in vivo, the two systems would be expected to be exquisitely sensitive to changes in concentration of their respective activating ligands.

Similar substrate recognition motifs for mammalian AMP-activated protein kinase, higher plant HMG-CoA reductase kinase-A, yeast SNF1, and mammalian calmodulin-dependent protein kinase I

We have analysed phosphorylation of the synthetic peptide AMARAASAAALARRR, and 23 variants, by mammalian, higher plant and yeast members of the SNF1 protein kinase subfamily (AMP‐activated protein kinase (AMPK), HMG‐CoA reductase kinase (HRK‐A), and SNF1 itself), and by mammalian calmodulin‐dependent protein kinase I (CaMKI). These four kinases recognize motifs which are very similar, although distinguishable. Our studies define the following recognition motifs: AMPK: Φ(X,β)XXS/TXXXΦ; HRK‐A: Φ(X,β)XXSXXXΦ; Snf1: ΦXRXXSXXXΦ; CaMKI: ΦXRXXS/TXXXΦ; where Φ is a hydrophobic residue (M, V, L, I or F) and β is a basic residue (R, K or H).

Human calcium-calmodulin dependent protein kinase I: cDNA cloning, domain structure and activation by phosphorylation at threonine-177 by calcium-calmodulin dependent protein kinase I kinase.

Human Ca(2+)‐calmodulin (CaM) dependent protein kinase I (CaMKI) encodes a 370 amino acid protein with a calculated M(r) of 41,337. The 1.5 kb CaMKI mRNA is expressed in many different human tissues and is the product of a single gene located on human chromosome 3. CaMKI 1–306, was unable to bind Ca(2+)‐CaM and was completely inactive thereby defining an essential component of the CaM‐binding domain to residues C‐terminal to 306. CaMKI 1–294 did not bind CaM but was fully active in the absence of Ca(2+)‐CaM, indicating that residues 295–306 are sufficient to maintain CaMKI in an auto‐inhibited state. CaMKI was phosphorylated on Thr177 and its activity enhanced approximately 25‐fold by CaMKI kinase in a Ca(2+)‐CaM dependent manner. Replacement of Thr177 with Ala or Asp prevented both phosphorylation and activation by CaMKI kinase and the latter replacement also led to partial activation in the absence of CaMKI kinase. Whereas CaMKI 1–306 was unresponsive to CaMKI kinase, the 1–294 mutant was phosphorylated and activated by CaMKI kinase in both the presence and absence of Ca(2+)‐CaM although at a faster rate in its presence. These results indicate that the auto‐inhibitory domain in CaMKI gates, in a Ca(2+)‐CaM dependent fashion, accessibility of both substrates to the substrate binding cleft and CaMKI kinase to Thr177. Additionally, CaMKI kinase responds directly to Ca(2+)‐CaM with increased activity.

Phosphorylation and Activation of Ca-Calmodulin-dependent Protein Kinase IV by Ca-Calmodulin-dependent Protein Kinase Ia Kinase PHOSPHORYLATION OF THREONINE 196 IS ESSENTIAL FOR ACTIVATION

Purified pig brain Ca-calmodulin (CaM)-dependent protein kinase Ia kinase (Lee, J. C., and Edelman, A. M.(1994) J. Biol. Chem. 269, 2158-2164) enhances, by up to 24-fold, the activity of recombinant CaM kinase IV in a reaction also requiring Ca-CaM and MgATP. The addition of brain extract, although capable of activating CaM kinase IV by itself, provides no further activation beyond that induced by purified CaM kinase Ia kinase, consistent with the lack of a requirement of additional components for activation. Activation is accompanied by the development of significant (38%) Ca-CaM-independent CaM kinase IV activity. In parallel fashion to its activation, CaM kinase IV is phosphorylated in a CaM kinase Ia kinase-, Ca-CaM-, and MgATP-dependent manner. Phosphorylation occurs on multiple serine and threonine residues with a Ser-P:Thr-P ratio of 3:1. The identical requirements for phosphorylation and activation and a linear relationship between extent of phosphorylation of CaM kinase IV and its activation state indicate that CaM kinase IV activation is induced by its phosphorylation. Replacement of Thr-196 of CaM kinase IV with a nonphosphorylatable alanine by site-directed mutagenesis abolishes both the phosphorylation and activation of CaM kinase IV, demonstrating that Thr-196 phosphorylation is essential for activation.

Phosphorylation of smooth muscle myosin by type II Ca2+/calmodulin-dependent protein kinase.

Brain type II Ca2+/calmodulin-dependent protein kinase was found to phoshorylate smooth muscle myosin, incorporating maximally ∼ 2 mol of phosphoryl per mol of myosin, exclusively on the 20,000 dalton light chain subunit. After maximal phosphorylation of myosin or the isolated 20,000 dalton light chain subunit by myosin light chain kinase, the addition of type II Ca2+/calmodulin-dependent protein kinase led to no further incorporation indicating the two kinases phosphorylated a common site. This conclusion was supported by two dimensional mapping of tryptic digests of myosin phosphorylated by the two kinases. By phosphoamino acid analysis the phosphorylated residue was identified as a serine. The phosphorylation by type II Ca 2+/calmodulin-dependent protein kinase of myosin resulted in enhancement of its actin-activated Mg2+-ATPase activity. Taken together, these data strongly support the conclusion that type II Ca2+/calmodulin-dependent protein kinase phosphorylates the same amino acid residue on the 20,000 dalton light chain subunit of smooth muscle myosin as is phosphorylated by myosin light chain kinase and suggest an alternative mechanism for the regulation of actin-myosin interaction.

Activation of a Calcium-Calmodulin-dependent Protein Kinase I Cascade in PC12 Cells

It has been observed that the activity of Ca2+-calmodulin (CaM)-dependent protein kinase I is enhanced up to 50-fold by its phosphorylation in vitro by a distinct CaM kinase I kinase (Lee, J. C., and Edelman, A. M. (1994) J. Biol. Chem. 269, 2158-2164). It has, however, been unclear whether this event represents an acute form of cellular regulation. We demonstrate here the phosphorylation and activation of CaM kinase I in PC12 pheochromocytoma cells in response to elevation of intracellular Ca2+. Treatment of PC12 cells with the Ca2+-ionophore, ionomycin, or with a depolarizing concentration of KCl, led to rapid, biphasic phosphorylation of CaM kinase I and to increases in CaM kinase I activity of 5.1- and 7.3-fold, respectively. Depolarization-induced activation of CaM kinase I was reduced by ∼80% by blockade of Ca2+ influx through L-type voltage-dependent Ca2+ channels and completely abolished by removal of extracellular Ca2+. The ability of PC12 cell CaM kinase I to be phosphorylated and activated by purified CaM kinase I kinase in vitro was markedly reduced by prior depolarization of the cells, consistent with intracellular phosphorylation and activation of CaM kinase I by CaM kinase I kinase. These results demonstrate the existence in PC12 cells of a CaM kinase I cascade, the function of which may be to sensitize cells to signal-induced elevations of intracellular Ca2+.

A Regulatory Feedback Loop Between Ca2+/Calmodulin-dependent Protein Kinase Kinase 2 (CaMKK2) and the Androgen Receptor in Prostate Cancer Progression

Background: Defining molecular mechanisms that regulate AR activity is critical for understanding prostate cancer progression. Results: CaMKK2 increases during disease progression, is transcriptionally regulated by the AR, promotes proliferation, and is required for optimal AR transcriptional activity. Conclusion: CaMKK2 is in a feedback circuit to maintain AR activity. Significance: The CaMKK2 pathway is a promising target for prostate cancer therapy. The androgen receptor (AR) plays a critical role in prostate cancer (PCa) progression, however, the molecular mechanisms by which the AR regulates cell proliferation in androgen-dependent and castration-resistant PCa are incompletely understood. We report that Ca2+/calmodulin-dependent kinase kinase 2 (CaMKK2) expression increases and becomes nuclear or perinuclear in advanced PCa. In the TRAMP (transgenic adenocarcinoma of mouse prostate) model of PCa, CaMKK2 expression increases with PCa progression with many cells exhibiting nuclear staining. CaMKK2 expression is higher in human castration-resistant tumor xenografts compared with androgen-responsive xenografts and is markedly higher in the AR-expressing, tumorigenic cell line LNCaP compared with cell lines that are AR-nonexpressing and/or nontumorigenic. In LNCaP cells, dihydrotestosterone induced CaMKK2 mRNA and protein expression and translocation of CaMKK2 to the nucleus. Conversely, androgen withdrawal suppressed CaMKK2 expression. Knockdown of CaMKK2 expression by RNAi reduced LNCaP cell proliferation and increased percentages of cells in G1 phase, whereas correspondingly reducing percentages in S phase, of the cell cycle. CaMKK2 knockdown reduced expression of the AR target gene prostate-specific antigen at both mRNA and protein levels, AR transcriptional activity driven by androgen responsive elements from the prostate-specific probasin gene promoter and levels of the AR-regulated cell cycle proteins, cyclin D1 and hyperphosphorylated Rb. Our results suggest that in PCa progression, CaMKK2 and the AR are in a feedback loop in which CaMKK2 is induced by the AR to maintain AR activity, AR-dependent cell cycle control, and continued cell proliferation.

Myosin light chain kinase is expressed in neurons and glia: immunoblotiing and immunocytochemical studies

The contractile protein myosin is thought to subserve motility-related functions in a wide range of eukaryotic non-muscle cells including both neurons and glia. To determine if the Ca2+/calmodulin-dependent enzyme, myosin light chain kinase (MLCK) is involved in the regulation of neural myosin we investigated the presence and localization of MLCK in a variety of neural tissues by immunoblotting and immunocytochemistry. A specific immunoreactive protein (M(r) = 146,000) was detected in blotted homogenates from many regions of rat brain and from primary cultures of either astrocytes or cerebellar granule cells grown in the absence of other cell types. At the light microscopic level, MLCK-immunoreactivity was evident in many regions of rat brain, as well as in the cultured astrocytes and cerebellar granule cells. MLCK-immunoreactivity was observed to be largely cytosolic in astrocytes but with a proportion associated with the cytoskeleton. In the cerebellar granule cells immunoreactivity was present in neuronal processes as well as somata. The detection of MLCK in neural cells suggests that MLCK-catalyzed myosin phosphorylation may couple changes in intracellular calcium concentrations to motility-related functions of neurons and glia.

Synthetic peptides based on the calmodulin-binding domain of myosin light chain kinase inhibit activation of other calmodulin-dependent enzymes

Nanomolar concentrations of synthetic peptides corresponding to the calmodulin-binding domain of skeletal muscle myosin light chain kinase were found to inhibit calmodulin activation of seven well-characterized calmodulin-dependent enzymes: brain 61 kDa cyclic nucleotide phosphodiesterase, brain adenylate cyclase, Bordetella pertussis adenylate cyclase, red blood cell membrane Ca++-pump ATPase, brain calmodulin-dependent protein phosphatase (calcineurin), skeletal muscle phosphorylase b kinase, and brain multifunctional Ca++ (calmodulin)-dependent protein kinase. Inhibition could be entirely overcome by the addition of excess calmodulin. Thus, the myosin light chain kinase peptides used in this study may be useful antagonists for studying calmodulin-dependent enzymes and processes.

Repression of Ca2+/calmodulin-dependent protein kinase IV signaling accelerates retinoic acid-induced differentiation of human neuroblastoma cells.

Neuroblastoma cells having stem cell-like qualities are widely employed models for the study of neural stem/progenitor cell proliferation and differentiation. We find that human BE(2)C neuroblastoma cells possess a signaling cascade initiated by Ca2+ influx via voltage-dependent calcium channels and the N-methyl-d-aspartate (NMDA) receptor and culminating in nuclear calmodulin-dependent protein kinase IV (CaMKIV)-mediated phosphorylation and activation of the transcription factors Ca2+/cyclic AMP-response element-binding protein (CREB) and ATF1 (activating transcription factor-1). This pathway functions to maintain BE(2)C cells in an undifferentiated, proliferative state. Parallel to this Ca2+-dependent pathway is a hormone-responsive program by which retinoic acid (RA) initiates the differentiation of BE(2)C cells toward a neuronal lineage. This is evidenced by RA-dependent induction of the cell cycle inhibitor p21/Cip1 (Cdk-interacting protein 1) and cell cycle arrest, induction of the neuroblastic marker doublecortin and of the neuron-specific intermediate filament protein, peripherin, and by RA-stimulated extension of neuritic processes. During neuronal differentiation there is a complex antagonistic interplay between these two major signaling pathways. RA down-regulates expression of CaMKIV and one of its upstream activators, CaMKK1 (calmodulin-dependent protein kinase kinase 1). This is accompanied by RA-induced suppression of activating phosphorylation of CREB with a time course paralleling that of CaMKIV down-regulation. RA-induced repression of the Ca2+/calmodulin-dependent protein kinase kinase/CaMKIV/CREB pathway appears to be involved in regulating the timing of neuronal differentiation, as shown by the effect of RNA interference of CaMKIV to markedly accelerate RA-dependent up-regulation of p21/Cip1 and doublecortin expression and RA-promoted neurite outgrowth. RA-induced repression of the CaMKIV signaling pathway may represent an early event in retinoid-dependent neuronal differentiation.