phosphorylate either a serine or a threonine side chain, and many are organized into clusters that seem to reflect their function-in transmembrane signal transduction, intracellular signal amplification, cell-cycle control, and so on.
磷酸化丝氨酸或苏氨酸侧链，许多被组织成似乎反映了它们在跨膜信号转导、细胞内信号放大、细胞周期控制等中的功能的簇。

As a result of the combined activities of protein kinases and protein phosphatases, the phosphate groups on proteins are continually turning over-being added and then rapidly removed. Such phosphorylation cycles may seem wasteful, but they are important in allowing the phosphorylated proteins to switch rapidly from one state to another: the more rapid the cycle, the faster a population of protein molecules can change its state of phosphorylation in response to a sudden change in its phosphorylation rate (see Figure 15-14). The energy required to drive this phosphorylation cycle is derived from the free energy of ATP hydrolysis, one molecule of which is consumed for each phosphorylation event.
由于蛋白激酶和蛋白磷酸酶的联合活性，蛋白质上的磷酸基团不断被过度添加，然后迅速去除。这种磷酸化循环可能看起来很浪费，但它们对于允许磷酸化的蛋白质从一种状态快速切换到另一种状态很重要：循环越快，蛋白质分子群可以越快地改变其磷酸化状态，以响应其磷酸化速率的突然变化（见图15-14）。驱动这种磷酸化循环所需的能量来自ATP水解的自由能，每个磷酸化事件消耗一个分子。

The hundreds of different protein kinases in a eukaryotic cell are organized into complex networks of signaling pathways that help to coordinate the cell's activities, drive the cell cycle, and relay signals into the cell from the cell's environment. Many of the extracellular signals involved need to be both integrated and amplified by the cell. Individual protein kinases (and other signaling proteins) serve as input-output devices, or "microprocessors," in the integration process. An important part of the input to these signal-processing proteins comes from the control that is exerted by phosphates added and removed from them by protein kinases and protein phosphatases, respectively.
真核细胞中的数百种不同的蛋白激酶被组织成复杂的信号通路网络，有助于协调细胞的活动，驱动细胞周期，并将信号从细胞环境中继到细胞中。许多涉及的细胞外信号需要被细胞整合和放大。单个蛋白激酶（和其他信号蛋白）在整合过程中充当输入输出设备或“微处理器”。这些信号处理蛋白输入的一个重要部分来自由蛋白激酶和蛋白磷酸酶分别从中添加和去除的磷酸盐所施加的控制。

The Src family of protein kinases (see Figure 3-10) exhibits such behavior. The Src protein (pronounced "sarc" and named for the type of tumor, a sarcoma, that its deregulation can cause) was the first tyrosine kinase to be discovered. It is now known to be part of a subfamily of nine very similar protein kinases, which are found only in multicellular animals. As indicated by the evolutionary tree in Figure , sequence comparisons suggest that tyrosine kinases as a group were a relatively late innovation that branched off from the serine/threonine kinases, with the Src subfamily being only one subgroup of the tyrosine kinases created in this way.
Src蛋白激酶家族（见图3-10）表现出这种行为。Src蛋白（发音为“sarc”，以其失调可能导致的肿瘤类型肉瘤命名）是第一个被发现的酪氨酸激酶。现在已知它是九种非常相似的蛋白激酶亚家族的一部分，这些激酶仅在多细胞动物中发现。如图 中的进化树所示，序列比较表明酪氨酸激酶作为一个群体是一个相对较晚的创新，从丝氨酸/苏氨酸激酶中分离出来，Src亚家族只是以这种方式产生的酪氨酸激酶的一个亚群。

The Src protein and its relatives contain a short -terminal region that becomes covalently linked to a strongly hydrophobic fatty acid, which anchors the kinase at the cytoplasmic face of the plasma membrane. Next along the linear sequence of
Src 蛋白及其亲戚包含一个短 末端区域，该区域与强疏水脂肪酸共价连接，该脂肪酸将激酶锚定在质膜的细胞质面上。接下来沿着线性序列
Figure 3-62 An evolutionary tree of selected protein kinases. A higher eukaryotic cell contains hundreds of such enzymes, and the human genome codes for more than 500 . Note that only some of these, those discussed in this book, are shown.
图 3-62 选定蛋白激酶的进化树。一个高等的真核细胞含有数百种这样的酶，而人类基因组编码的酶超过500种。请注意，本书中只显示了其中的一些内容。

amino acids come two peptide-binding domains, a Src homology 3 (SH3) domain and an SH2 domain, followed by the kinase catalytic domain (Figure 3-63). These kinases normally exist in an inactive conformation, in which a phosphorylated tyrosine near the C-terminus is bound to the domain, and the domain is bound to an internal peptide in a way that distorts the active site of the enzyme and helps to render it inactive.
氨基酸有两个肽结合结构域，一个 Src 同源性 3 （SH3） 结构域和一个 SH2 结构域，然后是激酶催化结构域（图 3-63）。这些激酶通常以无活性构象存在，其中 C 末端附近的磷酸化酪氨酸与 结构域结合，并且该 结构域以扭曲酶活性位点并有助于使其失活的方式与内部肽结合。

As shown in Figure 3-64, turning the kinase on involves at least two specific inputs: removal of the C-terminal phosphate and the binding of the SH3 domain by a specific activating protein. In this way, the activation of the Src kinase signals the completion of a particular set of separate upstream events (Figure 3-65). Thus, the Src family of protein kinases serves as specific signal integrators, contributing to the web of information-processing events that enable the cell to compute useful responses to a complex set of different conditions.
如图 3-64 所示，开启激酶至少涉及两个特定的输入：去除 C 端磷酸盐和通过特定激活蛋白结合 SH3 结构域。通过这种方式，Src激酶的激活标志着一组特定的独立上游事件的完成（图3-65）。因此，Src蛋白激酶家族作为特定的信号整合者，有助于信息处理事件网络，使细胞能够计算出对一组复杂不同条件的有用反应。

We have described how the addition or removal of phosphate groups on a protein can be used by a cell to control the protein's activity. In the example just discussed, a kinase transfers a phosphate from an ATP molecule to an amino acid side chain of a target protein. Eukaryotic cells also have another way to control protein activity by phosphate addition and removal. In this case, the phosphate is not attached directly to the protein; instead, it is a part of the guanine nucleotide GTP, which binds very tightly to a class of proteins known as GTP-binding proteins. In general, proteins regulated in this way are in their active conformations with GTP bound. The loss of a phosphate group occurs when the bound GTP is hydrolyzed to GDP in a reaction catalyzed by the protein itself, and in its GDP-bound state the protein is inactive. In this way, GTP-binding proteins act as on-off switches whose activity is determined by the presence or absence of an additional phosphate on a bound GDP molecule (Figure 3-66).
我们已经描述了细胞如何利用蛋白质上磷酸基团的添加或去除来控制蛋白质的活性。在刚才讨论的例子中，激酶将磷酸盐从ATP分子转移到靶蛋白的氨基酸侧链上。真核细胞还有另一种通过添加和去除磷酸盐来控制蛋白质活性的方法。在这种情况下，磷酸盐不直接附着在蛋白质上;相反，它是鸟嘌呤核苷酸 GTP 的一部分，它与一类称为 GTP 结合蛋白的蛋白质紧密结合。通常，以这种方式调节的蛋白质与GTP结合具有活性构象。当结合的 GTP 在蛋白质本身催化的反应中水解为 GDP 时，就会发生磷酸基团的丢失，并且在其 GDP 结合状态下，蛋白质是无活性的。通过这种方式，GTP 结合蛋白充当开关，其活性取决于结合的 GDP 分子上是否存在额外的磷酸盐（图 3-66）。

GTP-binding proteins (also called GTPases because of the GTP hydrolysis they catalyze) comprise a large family of proteins that all contain variations on the same GTP-binding globular domain. When a tightly bound GTP is hydrolyzed by the GTP-binding protein to GDP, this domain undergoes a conformational
GTP 结合蛋白（也称为 GTP 酶，因为它们催化的 GTP 水解）包含一大类蛋白质，这些蛋白质都包含同一 GTP 结合球状结构域的变体。当紧密结合的 GTP 被 GTP 结合蛋白水解为 GDP 时，该结构域发生构象
Figure 3-63 The domain structure of the Src family of protein kinases, mapped along the amino acid sequence. For the three-dimensional structure of , see Figure 3-13.
图 3-63 蛋白激酶 Src 家族的结构域结构，沿氨基酸序列绘制。的三维结构 见图3-13。

Figure 3-64 The activation of a Src-type protein kinase by two sequential events. As described in the text, the requirement for multiple upstream events to trigger these processes allows the kinase to serve as a signal integrator (Movie 3.11). (Adapted from S.C. Harrison et al., Cell . With permission from Elsevier.)
图 3-64 Src 型蛋白激酶通过两个连续事件激活。如文中所述，触发这些过程需要多个上游事件，这使得激酶可以充当信号积分器（电影 3.11）。（改编自 S.C. Harrison et al.， Cell .经爱思唯尔许可。

Figure 3-65 How a Src-type protein kinase acts as a signal-integrating device. A disruption of the inhibitory interaction illustrated for the SH3 domain (green) occurs when its binding to the indicated orange linker region is replaced with its higher-affinity binding to an activating ligand.
图 3-65 Src 型蛋白激酶如何充当信号整合装置。当 SH3 结构域（绿色）与指示的橙色连接子区域的结合被其与激活配体的高亲和力结合所取代时，就会发生抑制相互作用的破坏。

change that inactivates the protein. The three-dimensional structure of a prototypical member of this family, the monomeric GTPase called Ras, is shown in Figure .
使蛋白质失活的变化。该家族的原型成员，称为Ras的单体GTP酶的三维结构如图 所示。

The Ras protein has an important role in cell signaling (discussed in Chapter 15). In its GTP-bound form, it is active and stimulates a cascade of protein phosphorylations in the cell. Most of the time, however, the protein is in its inactive, GDP-bound form. It becomes active when it exchanges its GDP for a GTP molecule in response to extracellular signals, such as growth factors, that bind to receptors in the plasma membrane (see Figure 15-47).
Ras蛋白在细胞信号传导中起着重要作用（在第15章中讨论）。在其 GTP 结合形式中，它是活跃的并刺激细胞中蛋白质磷酸化的级联反应。然而，大多数时候，这种蛋白质处于非活性的、与GDP结合的形式。当它响应与质膜受体结合的细胞外信号（例如生长因子）将其 GDP 交换为 GTP 分子时，它就会变得活跃（见图 15-47）。

GTP-binding proteins are controlled by regulatory proteins that determine whether GTP or GDP is bound, just as phosphorylated proteins are turned on and off by protein kinases and protein phosphatases. Thus, Ras is inactivated by a GTPase-activating protein (GAP), which binds to the Ras protein and induces Ras to hydrolyze its bound GTP molecule to GDP-which remains tightly boundand inorganic phosphate , which is rapidly released. The Ras protein stays in its inactive, GDP-bound conformation until it encounters a guanine nucleotide exchange factor , which binds to GDP-Ras and causes Ras to release its GDP. Because the empty nucleotide-binding site is immediately filled by a GTP molecule (GTP is present in large excess over GDP in cells), the GEF activates Ras by indirectly adding back the phosphate removed by GTP hydrolysis. Thus, in a sense, the roles of GAP and GEF are analogous to those of a protein phosphatase and a protein kinase, respectively (Figure ).
GTP 结合蛋白由调节蛋白控制，调节蛋白决定 GTP 或 GDP 是否结合，就像磷酸化蛋白被蛋白激酶和蛋白磷酸酶打开和关闭一样。因此，Ras被GTP酶激活蛋白（GAP）灭活，该蛋白与Ras蛋白结合并诱导Ras将其结合的GTP分子水解为GDP-保持紧密结合的无机磷酸盐 ，并迅速释放。Ras蛋白保持其非活性的、与GDP结合的构象，直到它遇到鸟嘌呤核苷酸交换因子，该因子 与GDP-Ras结合并导致Ras释放其GDP。由于空的核苷酸结合位点立即被GTP分子填充（GTP在细胞中的含量远远超过GDP），GEF通过间接添加GTP水解去除的磷酸盐来激活Ras。因此，从某种意义上说，GAP和GEF的作用分别类似于蛋白磷酸酶和蛋白激酶的作用（图 ）。

Cells contain a special family of small proteins whose members are covalently attached to many other proteins to determine the activity or fate of the second protein. In each case, the carboxyl end of the small protein becomes linked to the amino group of a lysine side chain of a "target" protein through an isopeptide bond. The first such protein discovered, and the most abundantly used, is ubiquitin (Figure 3-69A). Ubiquitin can be covalently attached to target proteins in a variety of ways, each of which has a different meaning for cells. The major form of ubiquitin addition produces polyubiquitin chains in which-once the first ubiquitin molecule is attached to the target-each subsequent ubiquitin molecule links to Lys48 of the previous ubiquitin, creating a chain of Lys48-linked ubiquitins that are attached to a single lysine side chain of the target protein. This form of polyubiquitin directs the target protein to the interior of a proteasome, where it is digested to small peptides (see Figure 6-84). In other circumstances, only single molecules of ubiquitin are added to proteins. In addition, some target proteins are
细胞包含一个特殊的小蛋白家族，其成员与许多其他蛋白质共价连接，以确定第二种蛋白质的活性或命运。在每种情况下，小蛋白的羧基末端通过异肽键与“靶”蛋白的赖氨酸侧链的氨基相连。第一个发现的这种蛋白质，也是使用最广泛的蛋白质，是泛素（图3-69A）。泛素可以通过多种方式共价连接到靶蛋白上，每种方式对细胞都有不同的意义。泛素的主要形式产生多泛素链，其中 - 一旦第一个泛素分子连接到靶标 - 每个随后的泛素分子都连接到先前泛素的 Lys48，从而产生一条 Lys48 连接的泛素链，这些泛素连接到靶蛋白的单个赖氨酸侧链。这种形式的多泛素将靶蛋白引导到蛋白酶体内部，在那里它被消化成小肽（见图6-84）。在其他情况下，只有单个泛素分子被添加到蛋白质中。此外，一些靶蛋白是

Src-type protein kinase activity turns on fully only if the answers to all of the above questions are yes
只有当上述所有问题的答案都是肯定的时，Src 型蛋白激酶活性才会完全开启

OUTPUT 输出
Figure 3-66 GTP-binding proteins as molecular switches. The activity of a GTP-binding protein (also called a GTPase) generally requires the presence of a tightly bound GTP molecule (switch “on"). Hydrolysis of this GTP molecule by the GTP-binding protein produces GDP and inorganic phosphate ( ), and it causes the protein to convert to a different, usually inactive, conformation (switch "off"), Resetting the switch requires that the tightly bound GDP dissociates. This is a slow step that is greatly accelerated by specific signals; once the GDP has dissociated, a molecule of GTP is quickly rebound.
图 3-66 GTP结合蛋白作为分子开关。GTP结合蛋白（也称为GTP酶）的活性通常需要存在紧密结合的GTP分子（“打开”）。GTP 结合蛋白水解该 GTP 分子产生 GDP 和无机磷酸盐 （ ），并导致蛋白质转化为不同的、通常无活性的构象（“关闭”），重置开关需要紧密结合的 GDP 解离。这是一个缓慢的步骤，特定信号会大大加速;一旦GDP解离，GTP分子就会迅速反弹。
modified with a different type of polyubiquitin chain. These modifications have different functional consequences for the protein that is targeted (Figure 3-69B).
用不同类型的聚泛素链修饰。这些修饰对靶向蛋白质具有不同的功能后果（图3-69B）。

Related structures are created when a different member of the ubiquitin family, such as SUMO (small ubiquitin-related modifier), is covalently attached to a lysine side chain of target proteins. Not surprisingly, all such modifications are reversible. Cells contain sets of ubiquitylating and deubiquitylating (and sumoylating and desumoylating) enzymes that manipulate these covalent adducts, thereby playing roles analogous to the protein kinases and phosphatases that add and remove phosphates from protein side chains.
当泛素家族的不同成员（如 SUMO（小泛素相关修饰剂））共价连接到靶蛋白的赖氨酸侧链时，就会产生相关结构。毫不奇怪，所有这些修改都是可逆的。细胞含有一组泛素化和去泛素化（以及 sumoyating 和 desumoyation）酶，它们操纵这些共价加合物，从而发挥类似于从蛋白质侧链中添加和去除磷酸盐的蛋白激酶和磷酸酶的作用。

How do cells select target proteins for ubiquitin addition? As an initial step, the carboxyl end of ubiquitin needs to be activated. This activation is accomplished when a protein called a ubiquitin-activating enzyme (E1) uses ATP hydrolysis energy to attach ubiquitin to itself through a high-energy covalent bond (a thioester). E1 then passes this activated ubiquitin to one of a set of ubiquitin-conjugating (E2) enzymes, each of which acts in conjunction with a set of accessory (E3) proteins called ubiquitin ligases. There are roughly 30 structurally similar but distinct E2 enzymes in mammals, and hundreds of different E3 proteins that form complexes with specific E2 enzymes.
细胞如何选择泛素加法的靶蛋白？作为初始步骤，需要激活泛素的羧基末端。当一种称为泛素激活酶 （E1） 的蛋白质使用 ATP 水解能量通过高能共价键（硫酯）将泛素附着在自身上时，就会完成这种激活。然后，E1 将这种激活的泛素传递给一组泛素偶联 （E2） 酶之一，每种酶都与一组称为泛素连接酶的辅助 （E3） 蛋白结合作用。哺乳动物中大约有 30 种结构相似但不同的 E2 酶，以及数百种不同的 E3 蛋白与特定的 E2 酶形成复合物。

Figure 3-70 illustrates the process used to mark proteins for proteasomal degradation. [Similar mechanisms are used to attach ubiquitin (and SUMO) to other types of target proteins.] Here, the ubiquitin ligase binds to specific degradation signals, called degrons, in protein substrates, thereby helping E2 to form a polyubiquitin chain linked to a lysine of the substrate protein. This polyubiquitin chain on a target protein will then be recognized by a specific receptor in the proteasome, causing the target protein to be destroyed. Distinct ubiquitin ligases recognize different degradation signals, thereby targeting distinct subsets of intracellular proteins for destruction, often in response to specific signals (see Figure 6-86).
图 3-70 说明了用于标记蛋白质以进行蛋白酶体降解的过程。[类似的机制用于将泛素（和SUMO）连接到其他类型的靶蛋白上。在这里，泛素连接酶与蛋白质底物中的特定降解信号（称为degrons）结合，从而帮助E2形成与底物蛋白赖氨酸相连的多泛素链。然后，靶蛋白上的这种多泛素链将被蛋白酶体中的特异性受体识别，导致靶蛋白被破坏。不同的泛素连接酶识别不同的降解信号，从而靶向细胞内蛋白的不同亚群进行破坏，通常是对特定信号的反应（见图 6-86）。

SIGNALING BY PHOSPHORYLATED PROTEIN
磷酸化蛋白的信号传导

SIGNALING BY GTP-BINDING PROTEIN
通过GTP结合蛋白进行信号转导
Figure 3-68 A comparison of two major intracellular signaling mechanisms in eukaryotic cells. In both cases, a signaling protein is activated by the addition of a phosphate group and inactivated by the removal of this phosphate. Note that the addition of a phosphate to a protein can also be inhibitory. (Adapted from E.R. Kantrowitz and W.N. Lipscomb, Trends Biochem. Sci. .
图 3-68 真核细胞中两种主要细胞内信号转导机制的比较。在这两种情况下，信号转导蛋白通过添加磷酸基团而被激活，并通过去除该磷酸盐而失活。请注意，在蛋白质中添加磷酸盐也可能是抑制性的。（改编自 E.R. Kantrowitz 和 W.N. Lipscomb， Trends Biochem. Sci.

Figure 3-67 The structure of the Ras protein in its GTP-bound form. This monomeric GTPase illustrates the structure of a GTP-binding domain, which is present in a large family of GTP-binding proteins. The red regions change their conformation when the GTP molecule is hydrolyzed to GDP and inorganic phosphate by the protein; the GDP remains bound to the protein, while the inorganic phosphate is released. The special role of the "switch helix" in proteins related to Ras is explained in the text (see Figure 3-72 and Movie 15.7).
图 3-67 GTP 结合形式的 Ras 蛋白的结构。这种单体 GTP 酶说明了 GTP 结合结构域的结构，该结构域存在于一大家族的 GTP 结合蛋白中。当GTP分子被蛋白质水解为GDP和无机磷酸盐时，红色区域改变了它们的构象;GDP仍然与蛋白质结合，而无机磷酸盐被释放。文中解释了“开关螺旋”在与Ras相关的蛋白质中的特殊作用（见图3-72和电影15.7）。

(A)

(B)

The SCF ubiquitin ligase is a protein complex that binds different "target proteins" at different times in the cell cycle, covalently adding polyubiquitin polypeptide chains to these targets. Its C-shaped structure is formed from five protein subunits, the largest of which serves as a scaffold on which the rest of the complex is built. The structure underlies a remarkable mechanism (Figure 3-71). At one end of the is an E2 ubiquitin-conjugating enzyme. At the other end is a substrate-binding arm, a subunit known as an F-box protein. These two subunits are separated by a gap of about . When this protein complex is activated, the F-box protein binds to a specific site on a target protein, positioning the protein in the gap so that some of its lysine side chains contact the ubiquitin-conjugating enzyme. The enzyme can then catalyze repeated additions of ubiquitin polypeptide to these lysines (see Figure 3-71C), producing polyubiquitin chains that mark the target proteins for rapid destruction in a proteasome.
SCF泛素连接酶是一种蛋白质复合物，可在细胞周期的不同时间结合不同的“靶蛋白”，共价将聚泛素多肽链添加到这些靶点上。它的C形结构由五个蛋白质亚基组成，其中最大的亚基用作支架，复合物的其余部分就建立在支架上。该结构是一个非凡的机制的基础（图 3-71）。一端 是 E2 泛素偶联酶。另一端是底物结合臂，一个称为 F-box 蛋白的亚基。这两个亚基之间大约有 的间隙。当这种蛋白质复合物被激活时，F-box 蛋白与靶蛋白上的特定位点结合，将蛋白质定位在间隙中，使其一些赖氨酸侧链与泛素结合酶接触。然后，该酶可以催化泛素多肽的重复添加到这些赖氨酸中（见图3-71C），产生多泛素链，这些链标记靶蛋白，以便在蛋白酶体中快速破坏。

Figure 3-69 The marking of proteins by ubiquitin. (A) The three-dimensional structure of ubiquitin, a small protein of 76 amino acids. A family of special enzymes couples its carboxyl end to the amino group of a lysine side chain in a target protein molecule, forming an isopeptide bond. (B) Some modification patterns that have specific meanings to the cell. Note that the two types of polyubiquitylation differ in the way the ubiquitin molecules are linked together. Linkage through Lys48 signifies degradation by the proteasome (see Figure 6-84), whereas that through Lys63 has other meanings. Ubiquitin markings are "read" by proteins that specifically recognize each type of modification,
图3-69 泛素对蛋白质的标记。（A）泛素的三维结构，泛素是一种由76个氨基酸组成的小蛋白质。一个特殊酶家族将其羧基末端偶联到靶蛋白分子中赖氨酸侧链的氨基上，形成异肽键。（B）一些对细胞有特定含义的修饰模式。请注意，两种类型的多泛素化在泛素分子连接在一起的方式上有所不同。通过 Lys48 的键表示蛋白酶体的降解（见图 6-84），而通过 Lys63 的键具有其他含义。泛素标记被特异性识别每种修饰的蛋白质“读取”，

Figure 3-70 The marking of proteins with ubiquitin. (A) The C-terminus of ubiquitin is initially activated by being linked via a high-energy thioester bond to a cysteine side chain on the E1 protein. This reaction requires ATP, and it proceeds via a covalent AMP-ubiquitin intermediate. The activated ubiquitin on E1, also known as the ubiquitin-activating enzyme, is then transferred to the cysteine on an E2 molecule. (B) The addition of a polyubiquitin chain to a target protein. In a mammalian cell, there are several hundred distinct E2-E3 complexes. The E2s are called ubiquitinconjugating enzymes. The E3s are referred to as ubiquitin ligases. (Adapted from D.R. Knighton et al., Science 253:407-414, 1991.)
图3-70 用泛素标记蛋白质。（A） 泛素的 C 末端最初通过高能硫酯键与 E1 蛋白上的半胱氨酸侧链连接而被激活。该反应需要 ATP，并通过共价 AMP-泛素中间体进行。E1 上激活的泛素，也称为泛素激活酶，然后转移到 E2 分子上的半胱氨酸。（B）在靶蛋白上添加多泛素链。在哺乳动物细胞中，有数百种不同的 E2-E3 复合物。E2 被称为泛素结合酶。E3 被称为泛素连接酶。（改编自 D.R. Knighton 等人，《科学》253：407-414,1991 年。