05-364 Sigma-AldrichAnti-Cdk5 Antibody, clone DC17

Pituitary tumors are monoclonal adenomas that account for about 10-15% of intracranial tumors. Cyclin-dependent kinase 5 (CDK5) regulates the activities of various proteins and cellular processes in the nervous system, but its potential roles in pituitary adenomas are poorly understood. The kinase activity of CDK5 requires association with an activating protein, p35 (also known as CDK5 activator 1, p35). Here, we show that functional CDK5, associated with p35, is present in normal human pituitary and in pituitary tumors. Furthermore, p35 mRNA and protein levels were higher in pituitary adenomas than in the normal glands, suggesting that CDK5 activity might be upregulated in pituitary tumors. Inhibition of CDK5 activity in rat pituitary cells, reduced the expression of vascular endothelial growth factor (VEGF), a protein that regulates vasculogenesis and angiogenesis. Our results suggest that increased CDK5-mediated VEGF expression might play a crucial role in the development of pituitary adenomas, and that roscovitine and other CDK5 inhibitors could be useful as anticancer agents.

Retinoic acid (RA) has been believed to be an anticancer drug for a long history. However, the molecular mechanisms of RA actions on cancer cells remain diverse. In this study, the dose-dependent inhibition of RA on DU145 cell proliferation was identified. Interestingly, RA treatment triggered p35 cleavage (p25 formation) and Cdk5 overactivation, and all could be blocked by Calpain inhibitor, Calpeptin (CP). Subsequently, RA-triggered DU145 apoptosis detected by sub-G1 phase accumulation and Annexin V staining could also be blocked by CP treatment. Furthermore, RA-triggered caspase 3 activation and following Cdk5 over-activation were destroyed by treatments of both CP and Cdk5 knockdown. In conclusion, we report a new mechanism in which RA could cause apoptosis of androgen-independent prostate cancer cells through p35 cleavage and Cdk5 over-activation. This finding may contribute to constructing a clearer image of RA function and bring RA as a valuable chemoprevention agent for prostate cancer patients.

Increased levels of glutamate causing excitotoxic damage accompany neurological disorders such as ischemia/stroke, epilepsy and some neurodegenerative diseases. Cyclin-dependent kinase-5 (Cdk5) is important for synaptic plasticity and is deregulated in neurodegenerative diseases. However, the mechanisms by which kainic acid (KA)-induced excitotoxic damage involves Cdk5 in neuronal injury are not fully understood. In this work, we have thus studied involvement of Cdk5 in the KA-mediated degeneration of glutamatergic synapses in the rat hippocampus. KA induced degeneration of mossy fiber synapses and decreased glutamate receptor (GluR)6/7 and post-synaptic density protein 95 (PSD95) levels in rat hippocampus in vivo after intraventricular injection of KA. KA also increased the cleavage of Cdk5 regulatory protein p35, and Cdk5 phosphorylation in the hippocampus at 12?h after treatment. Studies with hippocampal neurons in?vitro showed a rapid decline in GluR6/7 and PSD95 levels after KA treatment with the breakdown of p35 protein and phosphorylation of Cdk5. These changes depended on an increase in calcium as shown by the chelators 1,2-bis(o-aminophenoxy)ethane-N,N,N?',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM) and glycol-bis (2-aminoethylether)-N,N,N?',N?'-tetra-acetic acid. Inhibition of Cdk5 using roscovitine or employing dominant-negative Cdk5 and Cdk5 silencing RNA constructs counteracted the decreases in GluR6/7 and PSD95 levels induced by KA in hippocampal neurons. The dominant-negative Cdk5 was also able to decrease neuronal degeneration induced by KA in cultured neurons. The results show that Cdk5 is essentially involved in the KA-mediated alterations in synaptic proteins and in cell degeneration in hippocampal neurons after an excitotoxic injury. Inhibition of pathways activated by Cdk5 may be beneficial for treatment of synaptic degeneration and excitotoxicity observed in various brain diseases.

Depression is one of the most pervasive and debilitating psychiatric diseases, and the molecular mechanisms underlying the pathophysiology of depression have not been elucidated. Cyclin-dependent kinase 5 (Cdk5) has been implicated in synaptic plasticity underlying learning, memory, and neuropsychiatric disorders. However, whether Cdk5 participates in the development of depressive diseases has not been examined. Using the chronic mild stress (CMS) procedure, we examined the effects of Cdk5/p35 activity in the hippocampus on depressive-like behaviour in rats. We found that CMS increased Cdk5 activity in the hippocampus, accompanied by translocation of neuronal-specific activator p35 from the cytosol to the membrane in the dentate gyrus (DG) subregion. Inhibition of Cdk5 in DG but not in the cornu ammonis 1 (CA1) or CA3 hippocampal subregions inhibited the development of depressive-like symptoms. Overexpression of p35 in DG blocked the antidepressant-like effect of venlafaxine in the CMS model. Moreover, the antidepressants venlafaxine and mirtazapine, but not the antipsychotic aripiprazole, reduced Cdk5 activity through the redistribution of p35 from the membrane to the cytosol in DG. Our results showed that the development of depressive-like behaviour is associated with increased Cdk5 activity in the hippocampus and that the Cdk5/p35 complex plays a key role in the regulation of depressive-like behaviour and antidepressant actions.

The roles of cyclin-dependent kinase 5 (Cdk5) in central nervous system and neurodegenerative diseases have been intensely investigated in recent decades. Because protein expressions of Cdk5 and its regulator, p35, have been identified in Leydig cells, it is informative to further explore the novel function of Cdk5/p35 in male reproduction. Here we show that Cdk5/p35 protein expression and kinase activity in mouse Leydig cells are regulated by human chorionic gonadotrophin (hCG) in both dose- and time-dependent manners. Blocking of Cdk5 by molecular inhibitors or small interfering RNA resulted in reduction of testosterone production by Leydig cells. cAMP, a second messenger in LH signaling, was identified as a factor in hCG-dependent regulation of Cdk5/p35. Importantly, Cdk5 protein and kinase activity could support accumulation of steroidogenic acute regulatory (StAR) protein, a crucial component of steroidogenesis. We additionally addressed the protein interaction between Cdk5/p35 and StAR. The Cdk5-dependent serine phosphorylation of StAR indicated a possible mechanism by which Cdk5 induced accumulation of StAR protein. In conclusion, Cdk5 modulates hCG-induced androgen production in mouse Leydig cells, possibly through regulation of StAR protein levels. These results indicate that Cdk5 may play an important role in male reproductive endocrinology and is a potential therapeutic target in androgen-related diseases.

Neuronal polarization requires coordinated regulation of membrane trafficking and cytoskeletal dynamics. Several signaling proteins are involved in neuronal polarization via modulation of cytoskeletal dynamics in neurites. However, very little is known about signaling proteins in the neuronal soma, which regulate polarized membrane trafficking and neuronal polarization. Protein kinase D (PKD) constitutes a family of serine/threonine-specific protein kinases and is important in regulating Golgi dynamics and membrane trafficking. Here, we show that two members of the PKD family, PKD1 and PKD2, are essential for the establishment and maintenance of neuronal polarity. Loss of function of PKD with inhibitor, dominant negative, and short interfering RNA disrupts polarized membrane trafficking and induces multiple axon formation. Gain of function of PKD can rescue the disruption of polarized membrane trafficking and neuronal polarity caused by cytochalasin D, which results in F-actin depolymerization. PKD1 and PKD2 are also found to be involved in the maintenance of neuronal polarity, as evidenced by the conversion of preexisting dendrites into axons on PKD inhibition. Unlike other polarity proteins, PKD does not interact with the cytoskeleton in neurites. Instead, PKD regulates neuronal polarity through its activity in the Golgi apparatus. These data reveal a novel mechanism regulating neuronal polarity in the Golgi apparatus.

The biological behaviors of thyroid cancer are varied, and the pathological mechanisms remain unclear. Some reports indicated an apparent aggregation of amyloid accompanying medullary thyroid carcinoma (MTC). Amyloid aggregation in neurodegeneration leads to hyperactivation of Cdk5 and subsequent neuronal death. Based on the connection with amyloid, the role of Cdk5 in MTC is worthy of investigation. Initially, the expression of Cdk5 and its activator, p35, in MTC cell lines was identified. Cdk5 inhibition by specific inhibitors or short interfering RNA decreased the proliferation of MTC cell lines, which reveals the importance of Cdk5 in MTC cell growth. Although p35 cleavage has been considered as an important element in neurodegeneration, it seems that p35 cleavage was not a major cause in Cdk5 activity-dependent MTC cell proliferation because neither Cdk5 activity nor cell growth was affected by the inhibition of p35 cleavage. Clearance of amyloid by antibody neutralization indicated that MTC cell proliferation was supported by calcitonin-derived extracellular amyloid and subsequent Her2 and Cdk5 activation. Significantly, the STAT3 pathway was involved in Cdk5-dependent proliferation of MTC cells through Ser-727 phosphorylation. In addition, Cdk5 inhibition reduced nuclear distributions of both the Cdk5-p35 complex and phospho-STAT3 in MTC cells. Finally, Cdk5 inhibition retarded tumor formation in vivo accompanying the reduction of phospho-STAT3. Our findings suggest the first demonstration of a novel and specific role for Cdk5 kinase in supporting the proliferation of the medullary thyroid carcinoma cells and could shed light on a new field for diagnosis and therapy of thyroid cancer.

Cyclin-dependent kinase 5 (Cdk5) is a unique member of the Cdk family; its kinase activity requires association with its activator, p35 or p39. p35 is the strongest and best characterized activator. Previous studies showed that p35 is a neuron-specific protein that restricts Cdk5 activity in neurons. However, a high expression level of Cdk5 is found in astrocytes, which raises the possibility that astrocytic Cdk5 is functional. Here we show the presence of functional Cdk5 associated with p35 in astrocytes and demonstrate its important role in process elongation of scratched astrocytes. We found that p35 and glial fibrillary acidic protein (GFAP) were co-localized in primary cultured and acute isolated brain cells. Cdk5 could form an immunocomplex with p35 and its activity was shown in pure primary cultured astrocytes. p35 was upregulated in astrocytes injured by scratching, concomitantly with upregulation of Cdk5 kinase activity. Pretreatment of the scratched astrocytes with a Cdk5 inhibitor, roscovitine, could delay wound healing by inhibiting the reorganization of tubulin, GFAP, and the extension of hypertrophic processes. Moreover, overexpression of dominant negative Cdk5 could shorten the length of extending protrusion of reactive astrocytes. Thus, our findings demonstrated that functional Cdk5, associated with p35, was expressed in astrocytes and its activity could be upregulated in reactive astrocytes, a new role of Cdk5 that has never been reported in the nervous system. The present study may provide new insight for understanding the multifunctional protein complex Cdk5/p35 in the nervous system.