Two Degradation Pathways of the p35 Cdk5 (Cyclin-dependent Kinase) Activation Subunit, Dependent and Independent of Ubiquitination

Abstract

Cdk5 is a versatile protein kinase that is involved in various neuronal activities, such as the migration of newborn neurons, neurite outgrowth, synaptic regulation, and neurodegenerative diseases. Cdk5 requires the p35 regulatory subunit for activation. Because Cdk5 is more abundantly expressed in neurons compared with p35, the p35 protein levels determine the kinase activity of Cdk5. p35 is a protein with a short half-life that is degraded by proteasomes. Although ubiquitination of p35 has been previously reported, the degradation mechanism of p35 is not yet known. Here, we intended to identify the ubiquitination site(s) in p35. Because p35 is myristoylated at the N-terminal glycine, the possible ubiquitination sites are the lysine residues in p35. We mutated all 23 Lys residues to Arg (p35 23R), but p35 23R was still rapidly degraded by proteasomes at a rate similar to wild-type p35. The degradation of p35 23R in primary neurons and the Cdk5 activation ability of p35 23R suggested the occurrence of ubiquitin-independent degradation of p35 in physiological conditions. We found that p35 has the amino acid sequence similar to the ubiquitin-independent degron in the NKX3.1 homeodomain transcription factor. An Ala mutation at Pro-247 in the degron-like sequence made p35 stable. These results suggest that p35 can be degraded by two degradation pathways: ubiquitin-dependent and ubiquitin-independent. The rapid degradation of p35 by two different methods would be a mechanism to suppress the production of p25, which overactivates Cdk5 to induce neuronal cell death. Cyclin-dependent kinases (Cdks)2 are a family of Ser/Thr kinases that are activated by binding a regulatory subunit called cyclin. Most members of Cdks are expressed in proliferating cells to promote cell cycle progression (1). In contrast, Cdk5 is activated by p35 or p39 non-cyclin proteins, which are mainly expressed in post-mitotic neurons (2). Cdk5 is a versatile kinase that is involved in many neuronal activities, including neuronal cell layer formation, synaptic transmission, membrane trafficking, and neuron cell death (3). p35 and p39 appear to share common and/or distinct functions for Cdk5, with p35 being the predominant activator. This is shown by the phenotypes of knock-out (KO) mice; p35 KO mice display abnormal neural layers in the cerebral cortex (4), and p39 KO mice do not show apparent abnormalities, whereas p35 and p39 double KO mice are perinatal lethal with abnormal neural layers, as are the Cdk5 KO mice (5-7). To understand the precise function of Cdk5-p35 in various neuronal activities, it is important to reveal the regulation mechanism of Cdk5 activity.As well as being cell cycle Cdks, Cdk5 is a stable protein and is expressed more abundantly than p35 in neurons (8, 9). Therefore, Cdk5 activity is determined primarily by the available amount of activator protein p35, and the protein amounts of p35 are regulated by the balance between synthesis and degradation (2). Although the synthesis of p35 is stimulated by NGF or BDNF (10, 11), the...

Tissue-type plasminogen activator regulates p35-mediated Cdk5 activation in the postsynaptic terminal

“…1E,F). Because p35 has a short half-life (Patrick et al, 1998), and its abundance in the synapse is controlled mainly by ubiquitindependent and -independent degradation pathways (Takasugi et al, 2016), and to a lesser extent by local protein synthesis (Patrick et al, 1998;Shah and Lahiri, 2014), we postulated that the rapid effect of tPA on p35 expression is due to inhibition of its degradation. To test this hypothesis, we first studied the effect of protein synthesis inhibition on the abundance of p35.…”

“…p35 is regulated transcriptionally by multiple factors, including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and tumor necrosis factor-alpha (TNFα) (Harada et al, 2001;Utreras et al, 2009); and postranscriptionally by several microRNAs (Moncini et al, 2017). Furthermore, its degradation is mediated by ubiquitin-dependent and -independent pathways (Takasugi et al, 2016).…”

Clinical role and biological function of CDK5 in hepatocellular carcinoma: A study based on immunohistochemistry, RNA-seq andin vitroinvestigation

“…Recent research revealed that P35 degradation occurs by both ubiquitin-dependent and ubiquitin-independent pathways. P35 degradation leads to the inhibition of P25 expression, which could over-activate CDK5 to induce neuronal cell death [ 27 ]. Various experiments demonstrated the significant role of CDK5 in the CNS by broadly disrupting in neuronal layering of various brain structures, such as the cerebral cortex, cerebellum, hippocampus and olfactory bulb [ 28 ].…”

Role of cyclin‐dependent kinases (CDKs) in hepatocellular carcinoma: Therapeutic potential of targeting the CDK signaling pathway

“…8 Cyclin-dependent kinase 5 was first recognized as a neuron-specific CDK due to the majority distribution of CDK5 activators residing within neurons. 16 However, emerging evidence shows CDK5 might have additional roles in gene expression, cell migration, apoptosis, and pathological processes, leading to inflammation, diabetes, immune dysfunction, and cancer [17][18][19][20][21][22] (Table 1).…”